Microbial nanowires

ABSTRACT

The application describes electrically conductive nanowires, as well as genetically and/or chemically modified nanowires with modified conductive, adhesive and/or coupling properties.

This application claims benefit of the priority filing dates of U.S.Patent Application Ser. No. 61/378,240, filed Aug. 30, 2010, and U.S.Patent Application No. 61/378,188, filed Aug. 30, 2010, the contents ofboth are specifically incorporated herein by reference in theirentireties.

This application is related to U.S. application Ser. No. ______, filedon Aug. 30, 2011 and entitled, “Microbial Nanowires and Methods ofMaking and Using Same,” which is incorporated by reference herein in itsentirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with support of the United States Governmentunder National Institute of Environmental Health Science SuperfundProgram Contract No. R01 ES017052-03 and National Science FoundationContract No. MCB-1021948. The Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Semiconductor electronics have exhibited a sustained exponentialdecrease in size and cost with a similar increase in performance overthe last thirty years. While such progress is expected to continue forseveral years, the economics and/or physical barriers of continued useof silicon for increasingly small and more powerful devices willultimately pose a challenge. For example, if only currently availabletechnologies are employed, the costs of building the necessarymanufacturing facilities will likely become prohibitive due to theshrinking size of devices, heat dissipation problems due to closelypacked structures, non-uniformity in dopant and conductive materials,and high electric fields that may lead to a cascade of breakdown eventswithin closely packed components.

Moreover, increases in pollution have been tied with increased energyconsumption for at least the last several hundred years. Acceleratedglobal warming and environmental degradation make the development ofalternative energy sources an urgent priority.

The world therefore needs new sources of energy and new materials foruse in fuel cells and nanoelectronic devices.

SUMMARY OF THE INVENTION

Microbes have the potential to address the problems of pollution, theneed for clean affordable energy and the need for new nanoelectronicmaterials. The invention described herein relates to microbial nanowiresthat conduct electricity. Such nanowires are made from microbial pilins(or pili). The invention also relates to expression cassettes,expression vectors and host cells (e.g., bacteria) that produce suchpilins and nanowires. In some embodiments, the nucleic acids encodingthe nanowire polypeptides are recombinantly modified so that nanowirepolypeptides with modified conductive properties can be produced.

Thus, one aspect of the invention is a nanowire polypeptide comprisingan amino acid sequence selected from the group consisting of SEQ IDNO:1-10. In some embodiments the amino acid sequence of the nanowirepolypeptide is genetically or chemically modified so that the nanowirepolypeptide has electrical conductivity activity that is different fromthe wild type nanowire polypeptide having any of SEQ ID NO: 1-10. Forexample, the electrical conductivity activity of the modified nanowirepolypeptide can be less than 90% or greater than 120% of the electricalconductivity activity of a wild type nanowire polypeptide comprising SEQID NO:1-10. In other embodiments, the modified nanowire polypeptideshave modified adhesive or coupling properties relative to wild-typenanowire polypeptides. In some embodiments, the nanowire polypeptide isan isolated polypeptide, meaning that it has been separated from itsnatural environment.

Another aspect of the invention is a pilus that includes such a modifiednanowire polypeptide. Further aspects of the invention include nucleicacids encoding such a genetically modified nanowire polypeptide,expression cassettes, expression vectors and host cells for expressingthe modified nanowire polypeptides.

DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows an alignment of the pilin nanowire sequences (SEQ IDNO:1-8) from the genomes of different Geobacteraceae species (top) and aprofile reflecting the residue conservation (bottom). Amino acidsconserved in all the sequences are marked with a star on top, thosesharing similar physicochemical characteristics are marked with a colon(‘:’), while those with somewhat different chemical or physicalcharacteristics are identified with a period or dot (‘.’).

FIG. 2A-C illustrates the primary and secondary structures of pili fromGeobacter sulfurreducens. FIG. 2A shows the SEQ ID NO:9 amino acidsequence of a nanowires pilin from Geobacter sulfurreducens. Thelocations of the conserved tyrosine residues (Y) are identified withblack shading while the negatively charged amino acids aspartic acid andglutamic acid (D and E, respectively) are identified with underliningand the positively charged amino acids lysine, arginine and histidine(K, R, and H, respectively) are identified with grey shading. FIG. 2Bshows the predominantly helical secondary structure of the Geobacterpilin nanowire (GEO) compared to that of the pilin of P. aeruginosastrain K (PAK), which serves as a structural model for other bacterialpili. FIG. 2C shows the location of certain amino acids and their sidechains within the structure of the Geobacter pilin nanowires.

FIGS. 3A-3B show that nanowire conductivity can be manipulated viagenetic engineering of the Geobacter pilin subunit. Pilin subunits wereengineered by using a nanowire polypeptide having an Y₅₇F replacement asstarting material (SEQ ID NO: 19). The replacement of tyrosine atposition 57 with phenylalanine removed one of the tyrosines used forelectron hopping along the nanowires. Single mutant (E₆₀A), doublemutant (D₅₃A, D₅₄A) and triple mutant (E₆₀A, D₅₃A, D₅₄A) polypeptideswere made where negatively charged amino acids were replaced withalanine residues at position 60, position 53 and/or position 54, asdescribed in the Examples. The negatively charged amino acids actnormally as proton acceptors of the tyrosines during electron transfer.As shown in FIG. 3A, the coulombic efficiencies, which depend on thecell's ability to metabolize the electron donor and convert it intoelectricity, were undisturbed. However, FIG. 3B shows that the aminoacid replacements resulted in nanowires with reduced conductivity, asindicated by the observed defects in the electron transfer rates toelectrodes in microbial fuel cells.

FIG. 4 illustrates electron transfer to Fe(III) oxides by cellsexpressing native (WT) nanowires or nanowires composed of pilinscarrying single (E60A), double (D53A D54A) and triple (E60A D53A D54A)replacements of negatively charged amino acids. The negatively chargedamino acids act as proton acceptors of the tyrosines during electrontransfer. Their replacement with alanine resulted in nanowires withreduced ability to reduce Fe(III) oxides.

FIG. 5A-C illustrate that the adhesive properties of nanowires can bemanipulated via genetic engineering. Nanowires were engineered with aS61A replacement to remove the glycosylation of serine at position 61.The replacement resulted in nanowires with reduced binding but sameconductivity, as indicated by the defect in Fe(III) oxide reduction(FIG. 5A), the maintenance of the coulombic efficiency (FIG. 5B) and theelectron transfer rates to electrodes (FIG. 5C) in microbial fuel cells.

FIGS. 6A-C illustrate the conductivity of purified wild-type Geobacterpili. FIGS. 6A and 6B show distal conductivity measurements of purifiedpili by scanning tunnel microscopy (STM). FIG. 6C shows axialconductivity along the purified pili of nanowires by ConductiveProbe-Atomic Force Microscopy (CP-AFM) according to an embodiment.

FIGS. 7A-C are images of wild-type G. sulfurreducens and P. aeruginosastrain K (PAK) pili. FIG. 7A is a STM topographical image of G.sulfurreducens pilus acquired using 0.5 V and 100 pA, while FIG. 7B is aSTM topographical image of PAK pili acquired at 3.0 V and 45 pA. FIG. 7Cshows STM I-V curves acquired at pili locations indicated by dots inFIG. 7A (black “A”) and FIG. 7B (gray “B”).

FIG. 8A-8C illustrate some of the structural features of G.sulfurreducens pili. FIG. 8A shows a TEM micrograph of negativelystained G. sulfurreducens pili purified as thick bundles at pH 7according to methods described in the Examples. FIG. 8B shows a TEMmicrograph of negatively stained G. sulfurreducens pili deaggregatedinto individual pilus filaments at pH 9.5. FIG. 8C shows asilver-stained gel (left panel) after SDS-PAGE ofoctyl-glucoside-depolymerized G. sulfurreducens pili, illustrating the6.5 kDa PilA protein band. The right panel of FIG. 8C illustrates thebinding of anti-PilA polyclonal antibodies with the G. sulfurreducenspili peptide.

FIG. 9 is a STM topographical image (top) with height measurements(bottom) of a section of the pilus fiber acquired in constant currentmode (0.5V, 100 pA).

FIGS. 10A and 10B illustrate the tunneling conductance, dI/dV, of G.sulfurreducens pili (FIG. 10A) and the P. aeruginosa strain K pilicontrols (FIG. 10B).

FIGS. 11A-C show spectra of G. sulfurreducens pili compared to variousstandards. FIG. 11A shows an absorption spectrum of purified G.sulfurreducens pili and, in the inset, the spectrum is shown incomparison to a standard. FIG. 11B shows a fluorescence spectrum ofpurified G. sulfurreducens pili, and in the inset, the fluorescentspectra of L-tyrosine (solid line) and menaquinone (dashed line). Notethat tyrosines yield two fluorescence peaks corresponding to thetyrosine (Tyr) and tyrosinate (Tyr•) forms. FIG. 11C shows afluorescence spectrum of a riboflavin standard solution in isopropanol.

FIGS. 12A-C illustrate measurement of I-V (current-voltage) curves inpurified pili. FIG. 12A is an AFM image of pili deposited onto a 25-nmthick gold electrode nanofabricated onto an insulating SiO₂ surfaceaccording to an embodiment. FIG. 12B is a schematic of a two-pointtransport measurement between the gold electrode and a CP-AFM tipthrough a pilus filament according to an embodiment. FIG. 12C show I-V(current-voltage) curves obtained with CP-AFM according to variousembodiments.

FIG. 13 shows a schematic density-of-states curve for an idealrectifier.

FIG. 14 is a STM topographical image (left) of a purified nanowiredeposited onto a graphite surface together with three spectroscopicmeasurements of the electronic density of states (right) according tovarious embodiments.

FIG. 15 shows a schematic of a likely charge path of the purifiednanowire of FIG. 14 (left) correlated with the three spectroscopicmeasurements shown at the right side of FIG. 14.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein relates to microbial nanowires withsequences modified to modulate their conductive, adhesive, couplingand/or other properties. Such nanowires are useful for development ofnanoelectronic devices and microbial fuel cells.

Microbial Nanowires

Geobacteraceae bacteria naturally produce protein filaments known aspili that are electrically conductive. For this reason, they aregenerally referred to as microbial or pilus nanowires. The pilusnanowires are protein filaments assembled on the cell envelope throughthe polymerization via hydrophobic interactions of a single peptidesubunit, the pilin or PilA. The purified pili are electricallyconductive. As the pili protrude outside the cell, other proteins, suchas metalloproteins known as c-cytochromes, can bind the pili and maycontribute to their conductivity and adhesive properties. However,biochemical analyses of the purified pilin subunits have demonstratedthat they were not directly associated with metals or metalloenzymeseven when they assemble into nanowires. Furthermore, they lack anybiological redox cofactors such as flavins and quinones. Thus, theconductivity of the pilin subunits is intrinsic to the nanowire proteinfilament and is not due to any redox-active component that may associatewith the nanowire polypeptide, such as metals, ions, contaminants,metalloenzymes, flavins or quinones.

The peptide subunit (or pilin) in the electrically conductive pili isencoded by the pilA gene of Geobacteraceae bacteria. The product of thepilA gene generates a peptide or PilA or pilin that polymerizes viahydrophobic interactions to form the pilus. The Geobacteraceae pilusnanowire electrically connects the cell with electron acceptors in itsenvironment. This electronic connection enables the cell to gain energythrough the transfer of metabolically-generated electrons acrosselectron transport proteins, such as c-cytochromes and othermetalloproteins of the cell envelope, and through the pilus. The pilusserves as the main electrical connection between the cell andextracellular acceptors such as Fe(III) oxides. Geobacter sulfurreducensis naturally found in underground sediment where anaerobic conditionsmay require that an electron acceptor other than oxygen be employed andwhere minerals or other electron acceptors are commonly available. Thus,although Geobacter sulfurreducens can utilize oxygen as an electronacceptor, these bacteria can also transfer electrons from their pili toextracellular electron acceptors such as Fe(III) oxides, resulting ininsoluble Fe(III) in the environment to be reduced to soluble Fe(II).

The pilus nanowires are dynamic filaments that protrude and retract bypolymerizing and depolymerizing the pilin subunits at the cell envelope.Thus, several pilin peptides are assembled to make a pilus that canfunction as a nanowire. Extension and retraction events are powered,respectively, by the PilB (pilin polymerase) and PilT (pilindepolymerase) proteins, which belong to the secretion NTPasesuperfamily. The pilus nanowires are predominantly helical (FIG. 2) instructure. In particular, they are composed of an α-helical corespanning the hydrophobic N-terminus region that promotes pilinpolymerization, and a short αβ-loop in the C-terminal region. Thus, theylack the long αβ-loop and extensive C-terminal globular head that otherbacterial pili possess.

Pilin assembly occurs via hydrophobic interactions proceeding in ahelical fashion that may help position electroactive amino acids bymerging or bonding their atomic orbitals optimally so as to favor chargetransport along and across the nanowire.

Examples of several amino acid sequences of nanowires pilins (or PilAsubunits) from different Geobacteraceae are shown in FIG. 1 (i.e., SEQID NO: 1-9).

Amino acids 20-90 of the Geobacter sulfurreducens PCA nanowire PilA withsequence accession number NP_(—)952547.1 (gi: 39996596) has thefollowing sequence (SEQ ID NO:1).

1 MIQKLRNRKG FTLIELLIVV AIIGILAAIA IPQFSAYRVK 41AYNSAASSDL RNLKTALESA FADDQTYPPE S

The Type IV pilin PilA from Geobacter sulfurreducens KN400 havingsequence accession number AD184335.1 (gi:298505612) has the followingsequence (SEQ ID NO:2).

1 MLQKLRNRKG FTLIELLIVV AIIGILAAIA IPQFSAYRVK 41AYNSAASSDL RNLKTALESA FADDQTYPPE S

The pilin domain-containing protein Geobacter lovleyi SZ having sequenceaccession number YP_(—)001952332.1 (gi:189425155) has the followingsequence (SEQ ID NO:3).

1 MLNKIRNRKG FTLIELLIVV AIIGILAAVA IPQFTTYRIK 41GYNSNATSDL RNLKTVLESV FADRQGYPGS

The pilin domain-containing protein of Pelobacter propionicus DSM 2379having sequence accession number YP_(—)901328.1 (gi: 118580078) has thefollowing sequence (SEQ ID NO:4).

1 MLNKLRNRKG FTLIELLIVV AIIGILAAIA IPQFSAYRAK 41AYNSAANSDL KNIKTGMEAF MADNQQYPGD VDYR

The domain from Geobacter metallireducens GS-15 having sequence homologyto Geobacter pilins and having accession number YP_(—)384358.1(gi:78222611) has the following sequence (SEQ ID NO:5).

1 MLQKLRNKKG FTLIELLIVV AIIGILAAIA IPQFAAYRQK 41AFNSAAESDL KNTKTNLESY YSEHQFYPN

The pilin from Geobacter sp. M21 having accession numberYP_(—)003021449.1 (gi:253700260) has the following sequence (SEQ IDNO:6).

1 MLNKLRSNKG FTLIELLIVV AIIGILAAIA IPQFSAYRAK 41AYNSAANSDL KNMKTGMEAY MADRQAYPAL LDQR

The pilin from Geobacter bemidjiensis Bem having accession numberYP_(—)002139394.1 (gi:197118967) has the following sequence (SEQ IDNO:7).

 1 MLNKLRSNKG FTLIELLIVV AIIGILAAIA IPQFSAYREK  41AYNAASNSDL KNFKTGLEAF NADFQTYPAA YVASTN

The pilin domain-containing protein from Geobacter sp. M18 havingaccession number ZP_(—)05310612.1 (gi:255058444) has the followingsequence (SEQ ID NO:8).

 1 MLNKIRSNKG FTLIELLIVV AIIGILAAIA IPQFSAYRAK 41AYNAAANSDL KNIKTGMEAY MADRQAYPVS LDER

FIG. 2 shows the SEQ ID NO:9 amino acid sequence of the pilin nanowiresubunit of Geobacter sulfurreducens. This SEQ ID NO:9 sequence isreproduced below.

 1 FTLIELLIVV AIIGILAAIA IPQFSAYRVK AYNSAASSDL 41RNLKTALESA FADDQTYPPE S

The amino acid sequence of the pilin PilA from Geobacter sulfurreducensis shown below as SEQ ID NO:10.

 1 MLQKLRNRKG FTLIELLIVV AIIGILAAIA IPQFSAYRVK 41AYNSAASSDL RNLKTALESA FADDQTYPPE SThe processing site for the SEQ ID NO: 10 signal peptide is between theglycine at position 10 and the phenylalanine at position 11. Removal ofthis signal peptide yields the SEQ ID NO:9 nanowire sequence shown inFIG. 2A. The N-terminal phenylalanine is also methylated duringprocessing of the signal peptide (not shown in FIG. 2A).

The SEQ ID NO:9 and 10 nanowire polypeptides are encoded by thefollowing pilA nucleic acid sequence (SEQ ID NO: 11).

  1 ATG CTT CAG AAA CTC AGA AAC AGG AAA GGT  31TTC ACC CTT ATC GAG CTG CTG ATC GTC GTT  61GCG ATC ATC GGT ATT CTC GCT GCA ATT GCG  91ATT CCG CAG TTC TCG GCG TAT CGT GTC AAG 121GCG TAC AAC AGC GCG GCG TCA AGC GAC TTG 151AGA AAC CTG AAG ACT GCT CTT GAG TCC GCA 181TTT GCT GAT GAT CAA ACC TAT CCG CCC GAA 211 AGT TAA

According to the invention microbial pilin subunits can be geneticallyengineered for controlled and/or customized electronic, microbial fueland other utilities. In one embodiment, the genetically engineeredmicrobial nanowire polypeptide is a modified Geobacter sulfurreducensnanowires polypeptide. In other embodiments, the genetically engineeredmicrobial nanowire polypeptide is a modification of any of the SEQ IDNO: 1-10 amino acid sequences. Such nanowire polypeptides can bemodified using available recombinant technology procedures to generatemutant nanowire polypeptides with modified conductive, adhesive,coupling and/or other properties.

The nanowires can include one or more subunits with various molecularweights (MW). The subunits can have a variety of molecular weightsranging from, for example, at least about 3-kDa, or higher, or betweenabout 3-kDa and about 25-kDa or between about 3-kDa and 20-kDa orbetween about 3-kDa and about 10-kDa or between about 4-kDa and about9-kDa, or between about 5.5-kDa and about 7.5-kDa, including any rangethere between. In one embodiment, the subunit molecular weight is about6.5-kDa or at least about 6.5-kDa. In one embodiment, nanowires formedby such subunits do not contain metals, ions, contaminants,metalloenzymes, flavins or quinones.

Thus, for example, any of the SEQ ID NO:1-10 amino acid sequences can bemodified using available recombinant technology procedures to generatemutant nanowire polypeptides with modified conductive, adhesive,coupling and/or other properties.

As illustrated herein, the tyrosine and charged amino acids are largelyresponsible for the conductive function of the nanowires. In someembodiments, tryptophan may also contribute to the conductive functionof the nanowires. Thus, to modulate the conductive function of thenanowires, their amino acid sequences can be modified to include agreater or lesser proportion of the tyrosine, tryptophan and/or chargedamino acids.

Amino acid residues of the nanowires can be genetically encoded L-aminoacids, naturally occurring non-genetically encoded L-amino acids,synthetic L-amino acids, D-enantiomers of any of the above andcombinations of any of these amino acids. The amino acid notations usedherein for the twenty genetically encoded L-amino acids and commonnon-encoded amino acids are conventional and are as shown in Table 1.

TABLE 1 Amino Acid One-Letter Symbol Abbreviation Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val β-Alanine bAla 2,3-Diaminopropionic Dpr acidα-Aminoisobutyric acid Aib N-Methylglycine MeGly (sarcosine) OrnithineOrn Citrulline Cit t-Butylalanine t-BuA t-Butylglycine t-BuGN-methylisoleucine MeIle Phenylglycine Phg Cyclohexylalanine ChaNorleucine Nle Naphthylalanine Nal Pyridylalanine 3-Benzothienyl alanine4-Chlorophenylalanine Phe(4-Cl) 2-Fluorophenylalanine Phe(2-F)3-Fluorophenylalanine Phe(3-F) 4-Fluorophenylalanine Phe(4-F)Penicillamine Pen 1,2,3,4-Tetrahydro- Tic isoquinoline-3- carboxylicacid β-2-thienylalanine Thi Methionine sulfoxide MSO Homoarginine hArgN-acetyl lysine AcLys 2,4-Diamino butyric Dbu acid P-AminophenylalaninePhe(pNH₂) N-methylvaline MeVal Homocysteine hCys Homoserine hSer E-Aminohexanoic acid Aha δ-Amino valeric acid Ava 2,3-Diaminobutyric Dab acid

Nanowire polypeptides that are encompassed within the scope of theinvention include any of these amino acids and include mutant nanowirepolypeptides having one or more of the amino acids within the SEQ ID NO:1-10 sequences substituted with other, different amino acids.

While the substituted or replaced amino acid may have similar physicaland chemical characteristics, it may also have different physical orchemical characteristics. For example, an amino acid from any of the SEQID NO:1-10 nanowire sequences that has no direct role in electricalconductivity may be replaced by an amino acid that has a direct role inelectrically conducting electrons along the pilus nanowire (e.g., atyrosine and/or a charged amino acid). Alternatively, an amino acid fromany of the SEQ ID NO: 1-10 nanowire sequences that has a direct role inelectrical conductivity may be replaced by an amino acid that has anindirect role in electrically conducting electrons along the pilusnanowires, or some other role such as adhesion, secondary or tertiarystructure formation, and the like.

In general, amino acids can be placed into three main classes:hydrophilic amino acids, hydrophobic amino acids and cysteine-like aminoacids, depending primarily on the characteristics of the amino acid sidechain. These main classes may be further divided into subclasses. Forexample, some types of hydrophobic amino acids have aromatic side chainswhile other types of hydrophobic amino acids do not have aromatic sidechains. Moreover, aromatic amino acids can have functional groups thatprovide a more hydrophilic character and that permit acceptance andtransport of electrons (e.g., tyrosine). In general, the hydrophilicand/or aromatic amino acids have a more direct role in the electricalconductivity functions of the pilus nanowires.

Hydrophilic amino acids include amino acids having acidic, basic oruncharged polar side chains and hydrophobic amino acids include aminoacids having apolar side chains. Apolar amino acids may be furthersubdivided to include, among others, aliphatic amino acids. Thedefinitions of the classes of amino acids as used herein are as follows:

“Hydrophobic amino acid” refers to an amino acid having a side chainthat is uncharged at physiological pH and that is repelled by aqueoussolution. Examples of genetically encoded hydrophobic amino acidsinclude Ala, Ile, Leu and Val. Examples of non-genetically encodedhydrophobic amino acids include t-BuA.

“Aromatic amino acid” refers to a hydrophobic or hydrophilic amino acidhaving a side chain containing at least one ring having a conjugatedπ-electron system (aromatic group). The aromatic group may be furthersubstituted with substituent groups such as alkyl, alkenyl, alkynyl,hydroxyl, sulfonyl, nitro and amino groups, as well as others. Examplesof genetically encoded aromatic amino acids include phenylalanine,tyrosine and tryptophan. Commonly encountered non-genetically encodedaromatic amino acids include phenylglycine, 2-naphthylalanine,β-2-thienylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and4-fluorophenylalanine.

“Apolar amino acid” refers to a hydrophobic amino acid having a sidechain that is generally uncharged at physiological pH and that is notpolar. Examples of genetically encoded apolar amino acids includeproline and methionine. Examples of non-encoded apolar amino acidsinclude Cha.

“Aliphatic amino acid” refers to an apolar amino acid having a saturatedor unsaturated straight chain, branched or cyclic hydrocarbon sidechain. Examples of genetically encoded aliphatic amino acids includeAla, Leu, Val and Ile. Examples of non-encoded aliphatic amino acidsinclude Nle.

“Hydrophilic amino acid” refers to an amino acid having a side chainthat is attracted by aqueous solution. Examples of genetically encodedhydrophilic amino acids include Ser and Lys. Examples of non-encodedhydrophilic amino acids include Cit and hCys.

“Acidic amino acid” or “negatively charged amino acid” refers to ahydrophilic amino acid having a side chain pK value of less than 7.Acidic amino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Examples of geneticallyencoded acidic amino acids include aspartic acid (aspartate) andglutamic acid (glutamate).

“Basic amino acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Examples of genetically encoded basic amino acidsinclude arginine, lysine and histidine. Examples of non-geneticallyencoded basic amino acids include the non-cyclic amino acids ornithine,2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.

“Polar amino acid” refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but where a bond in theside chain has a pair of electrons that are held more closely by one ofthe atoms involved in the bond. Examples of genetically encoded polaramino acids include asparagine and glutamine. Examples ofnon-genetically encoded polar amino acids include citrulline, N-acetyllysine and methionine sulfoxide.

“Cysteine-like amino acid” refers to an amino acid having a side chaincapable of forming a covalent linkage with a side chain of another aminoacid residue, such as a disulfide linkage. Typically, cysteine-likeamino acids generally have a side chain containing at least one thiol(SH) group. An example of a genetically encoded cysteine-like amino acidis cysteine. Examples of non-genetically encoded cysteine-like aminoacids include homocysteine and penicillamine.

As will be appreciated by those having skill in the art, the aboveclassifications are not absolute. Several amino acids exhibit more thanone characteristic property, and can therefore be included in more thanone category. For example, tyrosine has both an aromatic ring and apolar hydroxyl group. Thus, tyrosine has dual properties and can beincluded in both the aromatic and polar categories. Similarly, inaddition to being able to form disulfide linkages, cysteine also has anapolar character. Thus, while not strictly classified as a hydrophobicor an apolar amino acid, in many instances cysteine can be used toconfer hydrophobicity to a peptide.

Certain commonly encountered amino acids that are not geneticallyencoded and that can be present, or substituted for an amino acid, inthe peptides, peptide variants and peptide derivatives of the inventioninclude, but are not limited to, β-alanine (b-Ala) and other omega-aminoacids such as 3-aminopropionic acid (Dap), 2,3-diaminopropionic acid(Dpr), 4-aminobutyric acid and so forth; α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine(MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA);t-butylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (Phg);cyclohexylalanine (Cha); norleucine (Nle); 2-naphthylalanine (2-Nal);4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F));3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F));penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (MSO);homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid(Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNH₂));N-methyl valine (MeVal); homocysteine (hCys) and homoserine (hSer).These amino acids also fall into the categories defined above.

The classifications of the above-described genetically encoded andnon-encoded amino acids are summarized in Table 2, below. It is to beunderstood that Table 2 is for illustrative purposes only and does notpurport to be an exhaustive list of amino acid residues that maycomprise the peptides, variants and derivatives described herein. Otheramino acid residues that are useful for making the peptides, peptidevariants and peptide derivatives described herein can be found, e.g., inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Inc., and the references cited therein. Amino acidsnot specifically mentioned herein can be conveniently classified intothe above-described categories on the basis of known behavior and/ortheir characteristic chemical and/or physical properties as comparedwith amino acids specifically identified.

TABLE 2 Classification Genetically Encoded Genetically Non-EncodedHydrophobic Aromatic F, Y, W Phg, Nal, Thi, Tic, Phe(4- Cl), Phe(2-F),Phe(3-F), Phe(4-F), Pyridyl Ala, Benzothienyl Ala Apolar M, G, P ChaAliphatic A, V, L, I t-BuA, t-BuG, MeIle, Nle, MeVal, Cha, bAla, MeGly,Aib Hydrophilic Acidic D, E Basic H, K, R Dpr, Orn, hArg, Phe(p- NH₂),DBU, A₂ BU Polar Q, N, S, T, Y Cit, AcLys, MSO, hSer Cysteine-Like CPen, hCys, β-methyl-Cys

Nanowire peptides of the invention with modified conductive propertiescan have any amino acid replaced by tyrosine, tryptophan, or a chargedamino acid. Alternatively, nanowire peptides of the invention withmodified conductive properties can have any tyrosine, tryptophan, or acharged amino acid within the nanowire peptide replaced by another aminoacid.

Amino acid modifications that can diminish or abolish conductivityinclude single, double and triple replacements in tyrosines (e.g.,replaced with alanine or the structurally-similar phenylalanine as shownherein) and/or positively and negatively charged amino acids.

Amino acid modifications that can increase conductivity includereplacements that introduce additional tyrosines in optimum positionswithin the nanowire to promote electron transfer. Furthermore,replacements that result in structural changes that permit a moreoptimal electronic coupling between the electroactive amino acids (e.g.,by bringing closer together) can also be used because they may increasethe rates of electron hopping. These amino acids can be those directlyinvolved in the electron transfer, such as tyrosines, those serving asprotonating or proton-accepting residues, or those that preserve theoptimal nanowire structure to promote electron transfer.

In addition, amino acids carrying post-translational modifications suchas glycosylation, acylation or phosphorylation can be also introduced orreplaced to manipulate the binding and adhesive properties of thenanowires, the charge of the nanowires and the electronic behavior ofthe nanowires. Amino acids that are post-translationally modified can bereplaced, added, or used instead of an existing amino acid within any ofthe SEQ ID NO:1-10 peptides. For example, an amino acid subject toposttranslational modification, such as phosphorylation, glycosylationor acylation, can be used instead of an existing amino acid within anyof SEQ ID NO: 1-10. Alternatively, an amino acid that is notpost-translationally modified can be replaced with another amino acidthat is post-translationally modified. In some embodiments, the aminoacid is replaced with a similarly classified amino acid to minimizechanges in the secondary or tertiary structure of the nanowire peptide.

In some embodiments, a cysteine or cysteine-like amino acid is added toa nanowire peptide having a sequence like any of SEQ ID NO: 1-10.Alternatively, the cysteine or cysteine-like amino acid is used insteadof an amino acid present in a nanowire peptide having a sequence likeany of SEQ ID NO:1-10. Such a cysteine or cysteine-like amino acid isuseful for enhancing the binding or adhesion properties of the nanowirepeptide. For example, preliminary results indicate that placement oraddition of cysteine in nanowire peptides facilitates electricalcoupling of the nanowire peptide to substrates containing gold. In someembodiments, the cysteine or cysteine-like amino acid is placed withinor near the C-terminal region of the nanowire sequence.

In some embodiments, the nanowire polypeptides have a signal sequence;in other embodiments, the nanowire polypeptides do not have a signalsequence. As used herein, “signal sequence” or “signal peptide” refersto a peptide that can be used to secrete the heterologous polypeptideinto the periplasm of the bacteria. The signal for the heterologouspolypeptide may be homologous to the bacteria, or they may beheterologous, including signals native to the polypeptide being producedin the bacteria.

In some embodiments of the invention, the nanowire polypeptide isselected from the following group: (1) a polypeptide having an aminoacid sequence as shown in any of SEQ NO: 1-10; or (2) a polypeptidehaving an amino acid sequence with at least 70% identity to that of anyof SEQ ID NO: 1-10, the polypeptide having conductive function oractivity compared to that of the polypeptide of (1); or (3) a functionalfragment, variant, analog or derivative of the polypeptide of (1) or(2), having substantially the same biological function or activitycomparing to that of the polypeptide of (1) or (2). In some embodiments,the nanowire polypeptides contain an amino acid sequence with identityof at least 75%, or at least 80%, preferably at least 85%, preferably atleast 90%, preferably at least 95%, more preferably at least 96%, atleast 97%, at least 98% and/or at least 99% relative to any of SEQ NO:1-10.

As used herein, the term “polypeptide” refers to at least two amino acidresidues connected as a chain via covalent bonds such as peptide bonds,and can be recombinant polypeptides, natural polypeptides or syntheticpolypeptides. The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein.

The pilus nanowires are described further in the Examples. As describedherein, the purified pilus nanowire polypeptides, when stripped ofassociated proteins (such as c-cytochromes), metalloproteins, metals,ions, contaminants and redox cofactors such as flavins and quinones areelectrically conductive and behave as molecular rectifiers (asymmetricconductivity).

Manipulation of Nucleic Acids Encoding Nanowire Polypeptides

Nucleic acids encoding modified nanowire polypeptides are useful forrecombinant expression of the modified nanowire polypeptides. Nucleicacids encoding modified nanowire polypeptides can be generated fromnucleic acids encoding the naturally-occurring nanowire (e.g., pilA)nucleic acids using methods known to those of skilled in the art.

Any available nanowire nucleic acid can form the basis for generatingmutant nucleic acids that encode nanowires with modified properties. Forexample, Geobacteraceae bacteria naturally produce nanowire proteinfilaments that are electrically conductive. Hence, Geobacteraceaebacteria are one source of nanowire nucleic acids. Natural nucleic acidsequences, such those encoding the SEQ ID NO:1-10 nanowire polypeptides,can act as a basis for generating modified nanowire polypeptides.Naturally-occurring nanowire nucleic acid and amino acid sequences arealso available in public sequence databases such as those provided bythe National Center for Biotechnology Information (NCBI) database (see,e.g., the website at www.ncbi.nlm.nih.gov).

For example, the SEQ ID NO:9 and 10 nanowire polypeptides are encoded bythe following pilA nucleic acid sequence (SEQ ID NO: 11), which can beused to generate mutant nanowire nucleic acids.

  1 ATG CTT CAG AAA CTC AGA AAC AGG AAA GGT  31TTC ACC CTT ATC GAG CTG CTG ATC GTC GTT  61GCG ATC ATC GGT ATT CTC GCT GCA ATT GCG  91 ATT CCG CAG TTC TCG GCG TAT CGT GTC AAG 121GCG TAC AAC AGC GCG GCG TCA AGC GAC TTG 151AGA AAC CTG AAG ACT GCT CTT GAG TCC GCA 181TTT GCT GAT GAT CAA ACC TAT CCG CCC GAA 211 AGT TAA

In some of the embodiments, the nucleic acids that encode nanowirepolypeptides have sequence identity with the SEQ ID NO: 11 nucleic acidsequence of at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% and/orat least 99%.

For example, nucleic acids can readily be generated that encode mutantnanowire polypeptides, where in some embodiments, the mutant nucleicacids encode nanowire polypeptides that include less than the threetyrosine amino acids at positions 27, 32 and 57 of the SEQ ID NO:9nanowire polypeptide. Such ‘tyrosine-deficient’ nanowire polypeptideshave reduced conductivity, as illustrated herein. In other embodiments,the mutant nucleic acids encode nanowire polypeptides that include morethan the three tyrosine amino acid residues at amino acid positions 27,32 or 57. Such ‘tyrosine-rich’ nanowire polypeptides can have increasedconductivity. In some embodiments, the mutant nanowire polypeptides have2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 tyrosine residues.

In some embodiments, the mutant nucleic acids encode nanowirepolypeptides that include fewer negatively charged amino acids than aretypically present at positions 5, 39, 48, 53, 54 and 60 of the SEQ IDNO:9 nanowire polypeptide. In other embodiments, the mutant nucleicacids encode nanowire polypeptides that include more negatively chargedamino acids than are typically present at positions 5, 39, 48, 53, 54and 60 of the SEQ ID NO:9 nanowire polypeptide. Thus, the mutantnanowire polypeptides can, for example, have 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14 negatively charged amino acids (e.g., aspartic acid orglutamic acid).

In some embodiments, the mutant nucleic acids encode nanowirepolypeptides that include fewer positively charged amino acids than aretypically present at positions 28, 30, 41 and 44 of the SEQ ID NO:9nanowire polypeptide. In other embodiments, the mutant nucleic acidsencode nanowire polypeptides that include more positively charged aminoacids than are typically present at positions 28, 30, 41 and 44 of theSEQ ID NO:9 nanowire polypeptide. Thus, the mutant nanowire polypeptidescan, for example, have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14positively charged amino acids (e.g., aspartic acid or glutamic acid).

In many embodiments, the modified nanowire polypeptides recombinantlygenerated from the mutant nucleic acids have substantially the samesecondary and/or tertiary structure(s) as naturally occurring nanowirepolypeptides (e.g., the SEQ ID NO:1-10 polypeptides).

Methods for isolating nucleic acids encoding the naturally-occurringnanowires, as well as technologies for generation of nucleic acidsencoding modified nanowire polypeptides are available in the art. See,for example, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. eds.(John Wiley & Sons, Inc., 1999), or MOLECULAR CLONING: A LABORATORYMANUAL, Sambrook et al. (Cold Spring Harbor Laboratory Press, New York,1989), or MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al. (ColdSpring Harbor Laboratory Press, New York, 2001). Nucleic acids encodingmutant nanowire polypeptides containing various amino acid substitutionscan be produced by site-specific mutagenesis and polymerase chainreaction (PCR) amplification from the nucleic acids encoding thenaturally-occurring pilin. Stratagene provides a QuikChange mutagenesiskit that can be used for such mutagenesis. Complementary primerscontaining mutagenic nucleotides can be employed such as those describedin the Examples provided herein. Mutant nucleic acids that encode suchmodified nanowires can be produced, for example, by polymerase chainreaction (PCR) using primers that encode the desired sequence.

In some embodiments of the invention, the nanowire polypeptide isselected from the following group: (1) a polypeptide having an aminoacid sequence as shown in any of SEQ NO: 1-10; or (2) a polypeptidehaving an amino acid sequence with at least 40% sequence identity tothat of any of SEQ ID NO: 1-10, the polypeptide having conductivefunction or activity compared to that of the polypeptide of (1); or (3)a functional fragment, variant, analog or derivative of the polypeptideof (1) or (2), having substantially the same biological function oractivity comparing to that of the polypeptide of (1) or (2), wherein thepolynucleotides include: (a) polynucleotides that code the PilApolypeptides of (1), (2) or (3) above; (b) polynucleotides that arehybridized with, under low, medium or high stringent conditions, andhave at least 40% of sequence identity compared to the polynucleotidesof (a); and (c) polynucleotide fragments that contain polynucleotides asdescribed in (a) and (b).

The terms “stringent conditions” or “stringent hybridization conditions”include conditions under which a probe will hybridize to its targetsequence to a detectably greater degree than other sequences (e.g., atleast 2-fold over background). Stringent conditions are somewhatsequence-dependent and can vary in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which can be up to 100%complementarity to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). The probe can be approximately 20-500 nucleotides in length,but can vary greatly in length from about 18 nucleotides to equal to theentire length of the target sequence. In some embodiments, the probe isabout 10-50 nucleotides in length, or about 18-25 nucleotides in length,or about 18-50 nucleotides in length, or about 18-100 nucleotides inlength. In some embodiments, the probe is a full length nucleic acidwith SEQ ID NO: 11 (which has 216 nucleotides), or a fragment thereof.

Typically, stringent conditions will be those where the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide or Denhardt's.Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1M NaCl, 1% SDS (sodium dodecylsulfate) at 37° C., and a wash in 1×SSC to 2×SSC (where 20×SSC is 3.0 MNaCl, 0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderatestringency conditions include hybridization in 40 to 45% formamide, 1MNaCl, 1% SDS at 37° C., and a wash in 0.5×SSC to 1×SSC at 55 to 60° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65°C. Specificity is typically the function of post-hybridization washes,the critical factors being the ionic strength and temperature of thefinal wash solution. For DNA-DNA hybrids, the Tm can be approximatedfrom the equation of Meinkoth and Wahl (Anal. Biochem. 138:267-84(1984)):

T _(m)=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% formamide)−500/L

where M is the molarity of monovalent cations; % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % formamide is thepercentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. The Tm is reduced byabout 1° C. for each 1% of mismatching. Thus, the T_(m), hybridizationand/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with greater than or equalto 90% sequence identity are sought, the T_(m) can be decreased 10° C.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3or 4° C. lower than the thermal melting point (T_(m)). Moderatelystringent conditions can utilize a hybridization and/or a wash at 6, 7,8, 9 or 10° C. lower than the thermal melting point (T_(m)). Lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15 or 20° C. lower than the thermal melting point (T_(m)). Usingthe equation, hybridization and wash compositions, and desired T_(m),those of ordinary skill can identify and isolate nucleic acids withsequences related to SEQ ID NO:1. Those of skill in the art alsounderstand how to vary the hybridization and/or wash solutions. If thedesired degree of mismatching results in a Tm of less than 45° C.(aqueous solution) or 32° C. (formamide solution) it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inTijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY—HYBRIDIZATION WITH NUCLEIC ACID PROBES, part 1, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, N.Y. (1993); and CURRENT PROTOCOLS INMOLECULAR BIOLOGY, chapter 2, Ausubel, et al., eds, Greene Publishingand Wiley-Interscience, New York (1995). Unless otherwise stated, in thepresent application high stringency is defined as hybridization in4×SSC, 5×Denhardt's (5 g Ficoll, 5 g polyvinypyrrolidone, 5 g bovineserum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA,and 25 mM Na phosphate at 65° C., and a wash in 0.1×SSC, 0.1% SDS at 65°C.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or nucleic acids or polypeptides: (a)“reference sequence,” (b) “comparison window,” (c) “sequence identity,”(d) “percentage of sequence identity” and (e) “substantial identity.”

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. The reference sequence can be a nucleicacid sequence (e.g., SEQ ID NO: 11) or an amino acid sequence (e.g., anyof SEQ ID NO:1-10). A reference sequence may be a subset or the entiretyof a specified sequence; for example, as a segment of a full-length cDNAor genomic DNA sequence, or the complete cDNA or genomic DNA sequence,or a domain of a polypeptide sequence.

As used herein, “comparison window” refers to a contiguous and specifiedsegment of a nucleic acid or an amino acid sequence, wherein the nucleicacid/amino acid sequence may be compared to a reference sequence andwherein the portion of the nucleic acidamino acid sequence in thecomparison window may comprise additions or deletions (i.e., gaps)compared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The comparisonwindow can vary for nucleic acid and polypeptide sequences. Generally,for nucleic acids, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100 or morenucleotides. For amino acid sequences, the comparison window is at leastabout 15 amino acids, and can optionally be 20, 30, 40, 50, 100 or moreamino acids. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thenucleic acid or amino acid sequence a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of alignment of nucleotide and amino acid sequences forcomparison are well known in the art. The local homology algorithm(BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math 2:482, mayconduct optimal alignment of sequences for comparison; by the homologyalignment algorithm (GAP) of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-53; by the search for similarity method (Tfasta and Fasta) ofPearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA and TFASTA in the WisconsinGenetics Software Package, Version 8 (available from Genetics ComputerGroup (GCG™ programs (Accelrys, Inc., San Diego, Calif.)). The CLUSTALprogram is well described by Higgins and Sharp, (1988) Gene 73:237-44;Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et al., (1988) NucleicAcids Res. 16:10881-90; Huang, et al., (1992) Computer Applications inthe Biosciences 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.24:307-31. The preferred program to use for optimal global alignment ofmultiple sequences is PileUp (Feng and Doolittle, (1987) J. Mol. Evol.,25:351-60 which is similar to the method described by Higgins and Sharp,(1989) CABIOS 5:151-53 and hereby incorporated by reference). The BLASTfamily of programs which can be used for database similarity searchesincludes: BLASTN for nucleotide query sequences against nucleotidedatabase sequences; BLASTX for nucleotide query sequences againstprotein database sequences; BLASTP for protein query sequences againstprotein database sequences; TBLASTN for protein query sequences againstnucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel, et al., eds., GreenePublishing and Wiley-lnterscience, New York (1995).

GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-53, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package are 8 and 2, respectively. The gap creation and gapextension penalties can be expressed as an integer selected from thegroup of integers consisting of from 0 to 100. Thus, for example, thegap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity and Similarity. The Quality is the metric maximized in order toalign the sequences. Ratio is the quality divided by the number of basesin the shorter segment. Percent Identity is the percent of the symbolsthat actually match. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. The scoringmatrix used in Version 10 of the Wisconsin Genetics Software Package isBLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA89:10915).

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters (Altschul, et al., (1997) Nucleic Acids Res.25:3389-402).

As those of ordinary skill in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences, which may behomopolymeric tracts, short-period repeats, or regions enriched in oneor more amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen (1993) Comput. Chem. 17:149-63) and XNU (C.sub.1-ayerie andStates, (1993) Comput. Chem. 17:191-201) low-complexity filters can beemployed alone or in combination.

The terms “substantial identity” indicates that a polypeptide or nucleicacid comprises a sequence with between 55-100% sequence identity to areference sequence, preferably at least 55% sequence identity,preferably 60%, preferably 70%, more preferably 80%, most preferably atleast 90% or 95% sequence identity to the reference sequence over aspecified comparison window. The reference sequence can, for example, beany of the SEQ ID NO:1-10 nanowire polypeptides or the SEQ ID NO: 11nanowire nucleic acid. Optimal alignment may be ascertained or conductedusing the homology alignment algorithm of Needleman and Wunsch, supra.

Expression of Nanowire Polypeptides

Nucleic acids encoding nanowire polypeptides can be used for recombinantexpression of the nanowire polypeptides, for example, byoperably-linking the nanowire nucleic acid to an expression controlsequence within an expression vector, which can be introduced into ahost cell for expression of the encoded polypeptide.

As used herein, the term “operably linked” means that a nucleic acid andan expression control sequence are positioned in such a way that theexpression control sequence directs expression of the nucleic acid underappropriate culture conditions and when the appropriate molecules suchas RNA transcriptional proteins are bound to the expression controlsequence.

The term “expression control sequence” refers to a nucleic acid sequencesufficient to direct the transcription of another nucleic acid sequencethat is operably linked to the expression control sequence to produce anRNA transcript.

An “expression vector” is a nucleic acid molecule capable oftransporting and/or allowing for the expression of another nucleic acidto which it has been linked. Expression vectors contain appropriateexpression control sequences that direct expression of a nucleic acidthat is operably linked to the expression control sequence to produce atranscript. The product of that expression is referred to as a messengerribose nucleic acid (mRNA) transcript. The expression vector may alsoinclude other sequences such as enhancer sequences, synthetic introns,and polyadenylation and transcriptional termination sequences to improveor optimize expression of the nucleic acid encoding the nanowirepolypeptide.

The nanowire nucleic acid(s) can be optimized for expression in aselected prokaryotic (e.g., bacterial) or eukaryotic cell. As is knownto one of skill in the art, a particular type of bacterial or animalspecies may have a different set of preferred codons than another typeof species. Use of codons that are preferred by a host cell canfacilitate expression of the nanowire polypeptides. Optimized sequencesinclude sequences that are codon optimized to include codons that areemployed more frequently in one organism relative to another organism,as well as modifications to add or modify Kozak sequences, to add orremove introns, and/or to remove undesirable sequences, for instance,potential transcription factor binding sites.

In one embodiment, a nucleic acid sequence encoding nanowire isoptimized by replacing codons in a nanowire nucleic acid with codonsthat encode the same (or similar) amino acid but are preferentiallyemployed in a particular (selected) cell. Preferred codons have arelatively high codon usage frequency in a selected cell (e.g. abacterial, yeast or animal cell) and are translated more efficiently.Introduction of preferred codons can also result in the introduction ofonly selected transcription factor binding sites for transcriptionfactors present in the selected host cell, and relatively few otherundesirable structural attributes. Thus, the optimized nucleic acidproduct has an improved level of expression due to improved codon usagefrequency, and a reduced risk of inappropriate transcriptional behaviordue to a reduced number of undesirable transcription regulatorysequences.

In one embodiment, the optimized nucleic acid no longer hybridizes tothe corresponding non-optimized sequence, e.g., does not hybridize tothe non-optimized sequence under medium or high stringency conditions.However, in most embodiments, the optimized nucleic acid does hybridizeto the corresponding non-optimized sequence under medium or highstringency conditions. In another embodiment, the nucleic acid has lessthan 90%. e.g., less than 80%, nucleic acid sequence identity to thecorresponding non-optimized sequence and optionally encodes apolypeptide having at least 80%, e.g., at least 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or more, amino acid sequence identity with thepolypeptide encoded by the non-optimized sequence.

An isolated and optimized nucleic acid molecule of the invention mayhave a codon composition that differs from that of the correspondingwild type nucleic acid sequence at more than 30%, 35%, 40% or more than45%, e.g., 50%, 55%, 60% or more of the codons. For example, when anon-bacterial host cell is used, the preferred codons are those that areemployed more frequently in a selected non-bacterial host cell speciesthan, for example, in the genome of a Geobacter species. In general,preferred codons do not include codons that are infrequently used in theselected organism or cell type. Preferred codons for different organismsare known to the art, e.g., see www.kazusa.or.jp./codon/. In oneembodiment of the invention, the majority of the codons that differ areones that are preferred codons in a desired host cell.

A nucleic acid molecule encoding a nanowire polypeptide can optionallybe optimized for expression in a particular host cell and then operablylinked to one or more transcription regulatory sequences, e.g., apromoter, one or more enhancers, a transcription termination sequence ora combination thereof, to form an expression cassette.

Nucleic acids encoding nanowire polypeptides of the invention can beincorporated into bacterial, viral, insect, yeast or mammalianexpression vectors so that they are operably-linked to expressioncontrol sequences such as bacterial, viral, insect, yeast or mammalianpromoters (and/or enhancers).

Nucleic acid molecules or expression cassette that encode nanowirepolypeptides may be introduced to a vector, e.g., a plasmid or viralvector, which optionally includes a selectable marker gene, and thevector introduced to a cell of interest, for example, a bacterial, yeastor mammalian cell. In some embodiments, the vector may be maintained,manipulated, expanded or replicated in a prokaryotic cell such as a cellfrom the family Geobacteraceae or a cell from the genus Geobacter. Inother embodiments, the vector may be maintained, manipulated, expandedor replicated in a prokaryotic cell such as an E. coli, Streptomycesspp., Bacillus spp., Staphylococcus spp. and the like. In furtherembodiments, the vector may be maintained, manipulated, expanded orreplicated in a eukaryotic cell such as a yeast or mammalian cell. Insome preferred embodiments, the host cell is the bacterium Geobactersulfurreducens. Expression vectors containing nucleic acids encodingnanowire polypeptides can be introduced into bacterial, insect, yeast ormammalian host cells for expression using conventional methodsincluding, without limitation, transformation, transduction andtransfection. In some embodiments, the host cell also has a pilB and/orpilT gene, which may facilitate, respectively, assembly and extensionand/or retraction of the nanowire polypeptide(s). In other embodiments,the host cell has no pilT gene, or carries a deletion in the pilT gene,to inhibit retraction of the nanowire polypeptide(s) and facilitateassembly of the nanowire filament.

The expression of the encoded nanowire protein may be controlled by anypromoter capable of expression in prokaryotic cells or eukaryotic cells.Examples of prokaryotic promoters that can be used include, but are notlimited to, SP6, T7, T5, tac, bla, trp, gal, lac or maltose promoters.Examples of eukaryotic promoters that can be used include, but are notlimited to, constitutive promoters, e.g., viral promoters such as CMV,SV40 and RSV promoters, as well as regulatable promoters, e.g., aninducible or repressible promoter such as the tet promoter, the hsp70promoter and a synthetic promoter regulated by CRE. Vectors forbacterial expression include pGEX-5X-3, and for eukaryotic expressioninclude pCIneo-CMV. In some embodiments, the expression vector is thepRG5 vector (Coppi et al., Appl. Environ. Microbiol. 67: 3180-87(2001)); Leang et al., BMC Genomics 10, 331 (2009).

In many embodiments, the nanowire polypeptides are expressed in abacterial host cell. Plasmid vectors containing bacterial replicon andcontrol sequences are typically used for expression in a bacterial hostcell. The vector ordinarily carries a replication site, as well asmarking sequences that are capable of providing phenotypic selection intransformed cells. For example, E. coli is typically transformed usingpBR322, a plasmid derived from an E. coli species. See, e.g., Bolivar etal., Gene 2: 95 (1977). pBR322 contains genes conferring ampicillin andtetracycline resistance and thus provides an easy means for identifyingtransformed cells. The pBR322 plasmid, or other microbial plasmid orphage, also generally contains, or is modified to contain, promotersthat can be used by the bacterial organism for expression of theselectable marker genes.

Bacterial expression vectors for producing a nanowire polypeptide canalso contain an inducible promoter that is recognized by the hostbacterial organism and is operably linked to the nucleic acid encodingthe polypeptide of interest. It can also contain a separate promoter,which may be inducible or of low basal expression, operably linked tothe nucleic acid encoding the phage lysozyme. Inducible promoterssuitable for use with bacterial hosts include the β-lactamase andlactose promoter systems (Chang et al., Nature 275: 615 (1978); Goeddelet al., Nature 281: 544 (1979)), the arabinose promoter system,including the araBAD promoter (Guzman et al., J. Bacteriol. 174:7716-7728 (1992); Guzman et al., J. Bacteriol. 177: 4121-4130 (1995);Siegele and Hu Proc. Natl. Acad. Sci. USA, 94: 8168-8172 (1997)), therhamnose promoter (Haldimann et al., J. Bacteriol., 180: 1277-1286(1998)), the alkaline phosphatase promoter, a tryptophan (trp) promotersystem (Goeddel, Nucleic Acids Res., 8: 4057 (1980) and EP 36,776), theP_(LtetO-1) and P_(lac)/_(ara-1) promoters (Lutz and Bujard, NucleicAcids Res., 25: 1203-1210 (1997)), and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983)).However, other bacterial inducible and low-basal-expression promotersare suitable, including promoter nucleotide sequences that have beenpublished, thereby enabling a skilled worker operably to ligate them toDNA encoding the polypeptide of interest using linkers or adaptors tosupply any required restriction sites. For example, a strong and highlyleaky promoter, such as the trp promoter, can be employed. The phagelambda P_(L) promoter and/or the alkaline phosphatase promoter can alsobe used.

Promoters for use in bacterial systems also generally contain aShine-Dalgamo (S.D.) sequence operably linked to the DNA encoding thepolypeptide of interest. The promoter can be removed from the bacterialsource DNA by restriction enzyme digestion and inserted into the vectorcontaining the desired DNA. The phoA promoter can be removed from thebacterial-source DNA by restriction enzyme digestion and inserted intothe vector containing the desired DNA.

The nucleic acid encoding the nanowire polypeptide may contain a signalsequence, such as one at the N-terminus of the mature polypeptide. Thesignal sequence may be a component of the vector, or it may be a part ofthe polypeptide nucleic acid that is inserted into the vector. If aheterologous signal sequence is selected it should be one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell.

Expression vectors contain a nucleic acid sequence that enables thevector to replicate in one or more selected host cells. Such sequencesare well known for a variety of bacteria. The origin of replication fromthe plasmid pBR322 is suitable for most Gram-negative bacteria.

Expression vectors can also contain a selection gene, also termed aselectable marker. This gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for bacilli. One example of a selectionscheme utilizes a drug to arrest growth of a host cell. Those cells thatare successfully transformed with a heterologous gene produce a proteinconferring drug resistance and thus survive the selection regimen.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required. For analysis to confirmcorrect sequences in plasmids constructed, the ligation mixtures can betransformed into Geobacter, E. coli K12 strain 294 (e.g., ATCC 31,446),or other strains, and successful transformants are selected. Plasmidsfrom the transformants can be prepared, analyzed by restrictionendonuclease digestion, and/or sequenced by available procedures.

The nanowire nucleic acid molecule, expression cassette and/or vector ofthe can be introduced to a cell by any method including, but not limitedto, calcium-mediated transformation, electroporation, microinjection,lipofection, particle bombardment and the like.

Suitable bacteria for expression of the nanowire polypeptides includehost cells that also have or are modified to include a pilT gene. Suchhost cells include, for example, archeabacteria and bacteria, especiallyGram-negative bacteria. For example, Gram-negative bacteria such asGeobacteraceae or Enterobacteriaceae can be utilized as host cells.Examples of useful bacteria include Geobacter, Escherichia,Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella,Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, andParacoccus. Suitable E. coli hosts include E. coli W3110 (ATCC 27,325),E. coli 294 (ATCC 31,446), E. coli B, and E. coli X1776 (ATCC 31,537).These examples are illustrative rather than limiting. Mutant cells ofany of the above-mentioned bacteria may also be employed. It is, ofcourse, necessary to select the appropriate bacteria taking intoconsideration replicability of the replicon in the cells of a bacterium.For example, Geobacter, E. coli, Serratia, or Salmonella species can besuitably used as the host cells when well-known plasmids such as pBR322,pBR325, pACYC177, or pKN410 are used to supply the replicon.

The host cells containing the nanowire nucleic acid(s) can be expandedin culture using available procedures and cell culture conditions. Insome embodiments, pilus nanowires are produced by Geobacteraceaebacteria that have the pilB gene and the pilT gene (or, alternatively, adeletion of an endogenous pilT gene). The culture medium can be a FreshWater (FW) medium with acetate and fumarate, which is described byReguera et al. (NATURE 435: 1098-1101 (2005); Reguera et al., J.BACTERIOL. 189: 2125-27 (2007); U.S. Pat. No. 7,498,155, which areherein incorporated by reference in their entireties). The host cellscan be cultured at different temperatures, for example, at 20-30° C.(e.g., 25° C.).

Nanowire polypeptides can also be expressed in the absence of Fe(Ill)oxides when grown under suboptimal growth conditions analogous to thosethat Geobacteraceae would naturally encounter in environment. Piliproduction may not be specifically associated with the presence of metaloxides in their culture environment, but rather may be due to thephysiological state(s) associated with suboptimal growth, which occursat lower temperatures, during growth transitions, and whenGeobacteraceae have to use insoluble electron acceptors.

Expression of nanowires in the absence of Fe(III) oxides and atsuboptimal growth temperatures also causes the cells to agglutinate,indicating that the nanowires may participate in electron flow betweenthe cells. In experiments using Fe(III) oxide coated surfaces andelectrodes the inventors have demonstrated that G. sulfurreducens formsstructured biofilms and generates energy for growth by transferringelectrons across the biofilm cell layers, a process for which theexpression of the nanowires is needed. Pilus nanowires permit electroniccommunication between the biofilm cells and maintain the electronicefficiency per cell constant as the biofilm grows. The pili have astructural role in the biofilms and help maintain adequate cell spacingto provide optimum electronic communication and electron flow across thebiofilm.

The nanowires can be purified by any available method. In oneembodiment, the method comprises lysis of cells expressing thenanowires, followed by selective removal of contaminating cellmacromolecules, and then selective separation of pure nanowires fromother proteins. See Examples 5-7. In one embodiment, a single steppurification method is used which may have yields in excess of 50%, suchas up to 55% or up to 60% or higher, including any and all ranges therebetween. In one embodiment, the yield is at least about 63%. Higheryields, in excess of 63% may also be possible, such as up to about 95%,including any and all ranges there between. The protocol is flexible, inthe sense that it can be adapted for use with substantially any sampleof pili-expressing cells, substantially any method to removecontaminating cell macromolecules that do not affect the integrity ofthe nanowires, and substantially any method to selectively separate thenanowires from other contaminating proteins based on the nanowires'unique attributes.

The resulting nanowires are essentially pure, as they are stripped ofcontaminants, metals, ions, metalloenzymes, flavins, quinones and otherredox cofactors. In one embodiment, the purified nanowires are composedof a single peptide subunit (pilin or PilA) which polymerizes viahydrophobic interactions to form the pilus, i.e., nanowire filament.These nanowires can be stored dry substantially indefinitely and can beresuspended in appropriate solvents, as needed, for downstreamapplications. As noted above, surprisingly, these novel purifiednanowires have rectifying behavior due, in part, to the absence ofcellular contamination. Particular rectifying behavior is also due tothe protein composition (i.e., amino acid make-up) and structure of thenanowire.

In one embodiment, the rectifiers described herein are capable offunctioning as an asymmetric conductor for voltages of various ranges,such as, for example, voltages (V) having a range of about ±0.8 V or arange of about ±1.2 V. The rectifiers may also be useful at highervoltages. However, in some embodiments when higher voltages aregenerated, heat and/or damage to the rectifier and/or associatedmaterials may occur, reducing the performance of the one or morenanowires contained therein.

In one embodiment, the purified microbial nanowires function as one-wayconductors for voltages in the range of ±0.8 V (see the Examples).

The method provided by the invention for purifying nanowire polypeptideallows for the purification of nanowire polypeptides that properly foldto form the pilus structure described in the Examples.

Chemically Modified Nanowire Polypeptides

In some embodiments, the nanowire polypeptides are chemically modified.Such chemically modified nanowire polypeptides can be generated fromnanowire polypeptides with a natural (non-recombinantly engineered)sequence that is chemically modified. Alternatively, the chemicallymodified nanowire polypeptides can be a mutant nanowire polypeptide thatalso contains substitutions, deletions or additions of amino acids thatare not normally found in naturally occurring pilus nanowires. Thus, forexample, before chemical modification, the nanowire polypeptides canhave any of SEQ ID NO: 1-10, or a variant thereof. The nanowirepolypeptides can therefore have a genetically modified sequence made byany of the procedures described herein.

In some embodiments, the nanowire polypeptides can be chemicallymodified to modulate their conductive, adhesive, coupling and/or otherproperties. Such chemical modification can be performed by proceduresavailable in the art using a variety of reagents. For example, reagentssuch as performic acid, peroxides, iodoacetamide, iodoacetic acid,bissulfosuccinimidyl suberate, i-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-ethylmaleimide, methyl methanethiosulfonate andS-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methylmethanesulfonothioate (MTSL) can be used to modify the conductive,adhesive, coupling and/or other properties of the nanowire polypeptides.In other embodiments, the nanowire polypeptides can be glycosylated,acylated or conjugated to an alkylene glycol (e.g., polyethylene glycolor PEG). Such modifications can be performed by procedures available inthe art. See, e.g., John M. Walker, THE PROTEIN PROTOCOLS HANDBOOK(2002)(especially Part IV); Means, G. E. and Feeney, R. E. CHEMICALMODIFICATIONS OF PROTEINS. Holden-Day, San Francisco (1971).

Uses for the Nanowires

The nanowire polypeptides described herein have rectifying behavior dueto the protein composition (i.e., amino acid make-up) and structure ofthe nanowire. Such rectifying behavior is a characteristic of thepolypeptide in pure form, for example, in absence of metals and cellularcontaminants. The genetically and/or chemically modified nanowirepolypeptides also have rectifying behavior when in impure form, forexample, when present in vivo, in compositions and/or in articles ofmanufacture.

The term “rectifier” as used herein, refers to one or more nanoscalesolid-state electronic diodes that act as either a conductor or aninsulator, depending on the sign of the voltage. A rectifier providesasymmetric axial electronic conductance, i.e., differential forwardversus reverse conductivity. A single diode and rectifier essentiallyrefer to the same type of component. By combining more than one diodetogether, various properties of the rectifier can be altered.

The nanowire polypeptides can self-assemble into nanowires that caninclude one or more nanowire polypeptide subunits with various molecularweights (MW). The nanowire polypeptide subunits can have a variety ofmolecular weights ranging from, for example, at least about 3-kDa, orhigher, or between about 3-kDa and about 25-kDa or between about 3-kDaand 20-kDa or between about 3-kDa and about 10-kDa or between about4-kDa and about 9-kDa, or between about 5.5-kDa and about 7.5-kDa,including any range there between. In one embodiment, the subunitmolecular weight is about 6.5-kDa or at least about 6.5-kDa. In oneembodiment, such nanowires do not contain contaminants such as metals,ions, metalloenzymes, and redox cofactors such as flavins and quinones.

Rectifying devices using microbial nanowires are desirable because theycan be mass-produced and purified from recombinant hosts that aregenetically engineered to produce the nanowire subunit. The nanowirepolypeptide subunits can then be assembled in vitro or in vivo to formpili. This will enable the mass-production of microbial rectifiers at alow cost.

The rectifying behavior also opens, for example, the possibility toconstruct active devices such as transistors. With regard tonano-electronics, the rectifying behavior means that protein-baseddiodes (one-way conductors) can be constructed from these nanowires. Inconventional microelectronics, diodes are the basic building blocks fortransistors and more complex active components, including themicroprocessors that run our computers. Hence, in analogy, therectifying behavior of the nanowires opens the door to the constructionof protein-based nano-electronics transistors and more complex devices.

The most basic applications in nano-electronics include, for example,radio demodulation (rectification of AM radio frequency signals to makeaudio signals), low voltage AC-DC power conversion, current steering,power switches and over-voltage protection. More advanced applicationsinclude, but are not limited to, the logic circuitry in electronicdevices such as laptop computers, cellular phones and similar devices,further including computer chips, such as those used in thetransportation industry, such as in aircraft and automobiles.

In one embodiment, the purified microbial nanowires function as one-wayconductors for voltages in the range of ±0.8 V (see, e.g., Example 8).

In some embodiments, the nanowire polypeptides can be configured toinclude branches. Thus, the nanowire polypeptides can be assembled intoa main pilus that is elongated and has a selected or desirable length. Aplurality of branch pili may emanate from the main nanowire pilus at oneor more substantially fixed distances along the length of the mainpilus. The main pilus may also comprise one or more junctions with oneor more secondary main pili, where the junctions are substantiallyperpendicular to the length of the main pilus.

In another embodiment, the nanowire polypeptides can be configured toform part of an apparatus. For example, the apparatus may contain atleast one pilus comprising nanowires polypeptides. In other embodiments,the apparatus may contain at least one junction between pili. Forexample, the apparatus may include a plurality of junctions. Eachjunction may include a branch pilus and an elongate main pilus. Forexample, each junction may be situated at an interface between a branchpilus and the elongate main pilus.

As illustrated in the following non-limiting Examples, the inventorshave also demonstrated, for the first time, that chemical modification(e.g., which may include chemical stripping) and/or genetic engineering,can be used to manipulate the protein composition, structure and bindingproperties of microbial nanowires to selectively modify rectificationproperties. Microbial rectifiers also can be manipulated via geneticengineering to bind specific ligands for sensor design, controlled andspecific deposition during device manufacturing, etc.

Thus, the following non-limiting Examples further illustrate someaspects of the invention.

Example 1 Materials and Methods

This Example describes methods that can be used to make and analyzenanowire polypeptides.

Bacterial Growth Conditions.

Geobacter sulfurreducens strain PCA was used for the majority of thestudies of nanowire structure and function. Cells were routinely grownat 30° C. in NB medium (Coppi et al, APPL. ENVIRON. MICROBIOL. 67:3180-87 (2001)) supplemented with 15 mM acetate and 40 mM fumarate(NBAF) before being transferred three times in a modified fresh water(FW) medium (Lovley & Phillips, APPL. ENVIRON. MICROBIOL. 54: 1472-80(1988)) supplemented with 15 mM acetate and 40 mM fumarate (FWAF).Briefly, a concentrated (10×) basal FW medium stock containing NaHCO₃(25 g/L), NaH₂PO₄.H₂O (0.6 g/L), NH₄Cl (2.5 g/L), and KCl (1.0 g/L) wasprepared. The electron donor and acceptor were prepared as sterileconcentrated stocks (0.75 M sodium acetate and 1 M sodium fumarate,respectively) and the pH of the stock solutions was adjusted to 7 priorto autoclaving. Vitamins were prepared as separate solutions aspreviously described by Balch et al. (MICROBIOL. REV. 43: 260-96(1979)). Trace minerals were prepared as previously described by Lovleyet al. (APPL. ENVIRON. MICROBIOL. 48:81-87 (1984)), except that ZnSO₄was replaced with ZnCl₂ (0.13 g/L), and Na₂WO₄.2H₂O (0.025 g/L) wasadded. FWAF medium contained FW stock (96 ml/L), 0.75 M sodium acetate(20 ml/L), 1 M sodium fumarate (40 ml/L), vitamin solution (10 ml/L),mineral solution (10 ml/L) and ddH₂O to a final volume of 1 L. Themedium was dispensed in pressure tubes or serum bottles, sparged withN₂:CO₂ (80:20) to remove dissolved oxygen and sealed with butyl rubberstoppers and aluminum tear off seals (Wheaton) prior to autoclaving. Forpili induction, exponentially-growing cells from FWAF cultures grown at30° C. were subcultured in 100 ml of fresh FWAF with 30 mM acetate and40 mM fumarate and incubated at 25° C. until they reached earlystationary phase (ca. 72 h).

Isolation and Purification of Pili.

Pili were purified to homogeneity using a modification of a protocolpreviously used to purify enterobacterial fimbriae (see, Collinson etal. (J. BACTERIOL. 173: 4773-81 (1991)). Pili-expressing cells wereharvested by centrifugation (13,000×g) for 15 min at 25° C. andresuspended in 6 ml of 10 mM Tris HCl, pH 8.0 (Tris buffer; Invitrogen,99.9%). The cells were lysed by sonication (5 1-min at 4° C. per 1 ml ofcell suspension; Branson Sonifier 450) before adding RNase A (bovinepancreas; Roche Diagnostics) and DNase I (bovine pancreas. Sigma, 91%purity) enzymes to a 0.1 mg/ml final concentration and MgCl₂ (J. T.Baker, 99.4%) to a final concentration of 1 mM. The cell extracts wereincubated at 37° C. for 20 min to enzymatically degrade the nucleicacids in the sample. Lysozyme (hen egg white, Roche Diagnostics) wasthen added to a concentration of 1 mg/ml and incubated at 37° C. for 40min with gentle shaking (200 rpm, Innova 4340, New Brunswick). Cellmembranes and proteins in the extract were solubilized with sodiumdodecyl sulfate (SDS, 1% final concentration; Sigma 98.5%) afterincubation at 37° C. for 30 min. The SDS-insoluble fraction wascollected by centrifugation (12,100×g, 15 min, 25° C.) and washed twicewith 6 ml of Tris buffer. The SDS-insoluble fraction in Tris buffer wasdigested again with RNase, DNase and lysozyme, as described above.Mechanical vortexing (Fisher Scientific) was used to mix the samples.Samples with large clumps and/or aggregates were subjected to 2additional cycles of sonication for 1 min at 4° C. The insolublefraction was collected as described above, washed twice, and resuspendedin 1 ml of Tris buffer. When needed, the sample was stored at −20° C.overnight. The protein sample was suspended in 2 ml ofSDS-polyacrylamide gel electrophoresis (PAGE) sample buffer (10%[v/v]glycerol; 5% [v/v]b-mercaptocthanol; 2% [w/v] SDS, and 62.5 mM TrisHCl, pH 6.8) (Laemmli, Nature 227:680-85 (1970)) and boiled for 15 min.The SDS-treated sample was loaded on top of a preparative 12%polyacrylamide gel with a 5% stacking gel, and subjected toelectrophoresis at 40 mA for 5 h using Prep Cell 491 (Bio-Rad). Thematerial that did not enter the stacking gel was recovered by aspirationwith a pipette and washed three times with 1 ml ddH₂O of doubledistilled water by centrifugation (12,100×g, 15 min, 25° C.). Theprotein in the SDS-insoluble fraction was extracted twice with 95%ethanol (Decon Laboratories) and lyophilized or dried in a Speed Vacsystem (Savant Instruments Inc) at room temperature for approximately 20min. This ethanol step also solubilized organic contaminants such asquinone-like compounds or organic cofactors. The dried protein wasresuspended in 1 ml of ddH₂O and vortexed for 60 seconds to break up thelarge clumps. Poorly-bound protein in the insoluble material wasextracted with 0.2 M glycine (pH 1.5, adjusted with HCl; Invitrogen) at100° C. for 10 min. The insoluble fraction was recovered bycentrifugation (16,000×g, 25 min, 4° C.), washed five times with ddH₂O,and lyophilized or dried in a SpeedVac at room temperature untilcompletely dry. The dried sample was then stored at −20° C. forshort-term use or flash frozen in liquid nitrogen for long-term use.

Analytical Methods.

Quantitative elemental analysis of the purified pili preparations wasperformed by Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES) using a Thermo Jarrell-Ash Enviro 36 Inductively Coupled ArgonPlasm (Chemical Analysis Laboratory, University of Georgia, Athens). Forthese experiments, 1 ml aqueous samples of purified pili containing40-70 μg/ml of protein were analyzed in reference to blank controlsamples (without protein). When indicated, ethylenediaminetetraaceticacid (EDTA, Invitrogen) was added to the sample at a final concentrationof 0.1 mM prior to the ICP-AES analyses. Protein concentration wasdetermined using the bichinchoninic acid (BCA) assay (Pierce®, ThermoScientific; see Smith et al., Anal. Biochem. 150: 76-85 (1985)), withBovine Serum Albumin (BSA) as the protein standard.

Protein Electrophoresis.

Dried pili preparations were resuspended in 15 ml of ddH₂O containing 2%(w/v) Octyl-β-D-Glucopyranoside (OG) (Sigma, 98%) and incubated at roomtemperature for 1 h prior to SDS-PAGE. SDS-PAGE was performed accordingto the method of Laemmli as modified by Ames (Laemmli, Nature 227:680-85(1970); Ames, J. Biol. Chem. 249:634-44 (1974)). The OG-treated samplewas boiled in SDS-PAGE sample buffer (Laemmli, Nature 227:680-85 (1970))and subjected to electrophoresis on 12% ReadyGels (Bio-Rad) using a MiniProtean Tetra Cell apparatus (Bio-Rad). After electrophoresis, the gelswere silver stained using the Pierces Silver Stain for Mass Spectrometrykit (Thermo Scientific), following the instructions supplied bymanufacturer. After silver-staining, the ca. 6.5-kDa PilA protein bandwas excised from the gel, destained and digested with trypsin followingthe procedure described by manufacturer (Pierce® Silver Stain for MassSpectrometry, Thermo Scientific). The peptides in the tryptic digestwere concentrated and purified chromatographically with Cisreversed-phase media (ZipTip®, Millipore) and separated by matrixassisted laser desorption ionization-time of flight spectrometry(MALDI-TOF, Shimadzu Axima). Peptide identification and prediction ofpotential contributions of post-translational modifications to thepeptide mass was performed using the MS-DIGEST tool at theProteinProspector database from the University of California, SanFrancisco (see website atprospector.ucsf.edu/prospector/cgi-binimsform.cgi?form=msdigest).

Western Blot (Immunoblot) Analysis.

Proteins separated by SDS-PAGE were electrophoretically transferred to anitrocellulose membrane (HyBond ECL™, Amersham GE Healthcare) at 50 Vfor 15 min using a Mini Protean Tetra Cell apparatus (Bio-Rad). Therapid western blotting kit (Amresco®) was used for the electrophoretictransfer and membrane blocking, following manufacturer'srecommendations. After blocking, the membrane was incubated in 10 mlrapid antibody diluent solution, (45 min, room temperature, gentleagitation) with a 1:5000 dilution of the primary antibody (rabbit α-PilApolyclonal antibodies raised against the 42 amino acids at thecarboxy-terminus of the PilA protein) and a 1:2500 dilution of goatα-rabbit IgG antibodies conjugated to the Cy^(tm) 5 fluorescence dye(ECL™ Plex, Amersham GE Healthcare). The membrane was washed in rapidwash solution provided by manufacturer (3 times 5 min). The membrane wasthen scanned with Typhoon imager operated in fluorescence mode(excitation at 633 nm, 670 BP 30 filter, and PMT setting at 600 V) tovisualize the protein bands that hybridized with the primary antibodies.

Microscopy.

For Confocal Laser Scanning Microscopy (CLSM), dried preparations ofpurified pili were dissolved in phosphate buffer saline (PBS), depositedon the surface of a glass cover slip and allowed to adsorb for 30 min.The adsorbed pili were then washed with PBS and fixed with 100 μl of3.7% paraformaldehyde in PBS. After washing with PBS, the samples wereincubated for 30 min in PBS containing 1% BSA, before adding theanti-PilA primary antibody (1:100) and incubating at 4° C. overnight.Following three washes in PBS-1% BSA, the samples were incubated withthe secondary antibody (α-rabbit conjugated to Alexa fluor 488 dye,1:1000) for 1 h. The cover slip was then washed 3 times with PBS bufferand examined with Zeiss LSM Pascal confocal microscope equipped with aPlan-Neofluar 63x oil objective (excitation, 488 nm; emission, 505-535nm). For transmission electron microscopy (TEM), an aqueous solution ofpurified pili was adsorbed on a carbon-copper grid (Mesh 300, ElectronMicroscopy Sciences), negatively stained with 1% (w/v) uranyl acetate indistilled water. The negatively stained samples were examined with aJeol 100 CX electron microscope (Japan Electron Optic Laboratory)operated at 100 kV.

Scanning Probe Microscopy.

Distal (lateral) and axial (length) conductivity measurements wereperformed, respectively, by scanning tunneling microscopy (STM) andconductive probe-atomic force microscopy (CP-AFM). STM imaging andspectroscopy was performed as described by Veazey et al. (Filament-likegraphite artifacts by STM, ULTRAMICROSCOPY (2010)). For CP-AFMmeasurements a Bio-AFM-CF instrument (Asylum) was used and a goldelectrode grid nanofabricated onto a silicon substrate was used forbiological deposition. For the fabrication of the gold grid, photoresist(Shipley S1813) was spin-coated onto silicon wafers having a 300 nmthermal oxide layer (SiO₂). After photoresist development, patternedgold electrodes were deposited by thermally evaporating 5 nm of titaniumfollowed by 25 nm of gold onto the surface of the wafer. A solutioncontaining ca. 40-70 μg/ml of purified pili in ddH₂O were then depositedonto the electrodes, left to adsorb for 25 minutes, and then wicked drywith absorbent paper. CP-AFM was performed with Pt-coated cantilevershaving spring constant 2 N/m (Veeco). Pilus nanowires lying across thegold-SiO₂ interface were first identified in imaging mode. For currentvoltage (I-V) measurements, the tip was placed on a point of the piluslying on the SiO₂. Positive controls were generated by positioning thetip on the gold electrode, while negative controls were produced bypositioning the tip on the SiO₂ substrate at 100-nm distances from thegold edge.

Example 2 Conservation of Geobacteraceae pilA Amino Acid Sequences andStructures

The amino acid sequences encoded by the pilA gene of severalGeobacteraceae were examined to identify conserved amino acids in thepilA nanowire gene product. Alignment of all the pilin-like sequences inthe available Geobacteraceae genomic sequences demonstrated that threetyrosines are conserved in all the Geobacteraceae pilins, suggesting itis a key residue for the nanowire function (FIG. 1). Furthermore,conserved positively charged (arginine and lysine) amino acids arepositioned in the vicinity of some of the tyrosines, a commonarrangement that keeps the aromatic orbitals empty and available toreceive an electron, thus promoting electron hopping (FIG. 2). On theother hand, conserved negatively-charged amino acids are also positionedin the vicinity of some of the tyrosines (FIG. 2A) and may serve asproton acceptors to facilitate the electron hopping.

Geobacter pilins belong to the subclass type IVa, which is broadlydefined by their short (ca. 150 amino acids) length and conserved N-tamino acid sequence carrying the recognition site for a dedicatedpre-pilin peptidase. In addition, all type IVa pilins carry anN-terminal N-methylated phenylalanine upon processing. The Geobacterpilins, though much shorter than other type IVa pilins (˜60-70 aminoacids), contain the conserved N-terminal sequence required forprocessing and assembly and the N-methylated phenylalanine of type IVapilins. However, amino acid divergence at the C-terminus places them inan independent line of descent.

The secondary structure of the Geobacter pilin also is unique among allknown type IVa pilins (FIG. 2B). It contains the hydrophobic N-terminalα-helix that promotes pilin polymerization, a short αβ-loop, and lacksthe C-terminal globular head (with a disulfide bond) that confers on thepilus its specific functions. Particular structures, in particularα-helix conformations, have been shown to contribute to peptideconductivity. Furthermore, the pilin of P. aeruginosa strain K (PAKpilin), which serves as a structural model for type IVa pilins, has theC-terminal globular head and polymerizes in vivo to producenonconductive pilus filaments.

Pilin assembly via hydrophobic interactions proceeds in a helicalfashion and may help position electroactive amino acids and merge orbond their atomic orbitals optimally so as to favor charge transportalong and across the nanowire.

Example 3 Genetic Manipulation of Redox-Active Amino Acids in PilinNanowires

This Example illustrates that manipulation of redox-active amino acidsin the pilin nanowires enables the generation of nanowires with alteredconductive properties.

Previous work has indicated that aromatic amino acids are redox-activeamino acids that serve as electronic mediators of protein electrontransfer. When properly positioned in close proximity to each other theyfunction as redox-active aromatic intermediates and create an electronhopping pathway. Preliminary evidence indicates that electron hopping isfavored by amino acids that can be easily oxidized such as tyrosines andtryptophans.

Since the rates of electron hopping are linearly dependent on thedistance between relay amino acids, site-directed mutagenesis was usedto genetically manipulate the distance between tyrosine residues in thepilus. As a proof of concept, tyrosine Y₅₇ was initially replaced withan alanine (Y₅₇A) and later with a phenylalanine (Y--F). The tyrosine atposition 57 (Y₅₇) was a good candidate because it is at the C-terminalend of the pilin and exposed to the pilus exterior, so its replacementdid not radically affect the pilus structure. Alanine was initially usedto replace the Y₅₇ residue because it clearly has no redox activity.Phenylalanine was later used to replace the Y₅₇ residue because it isstructurally similar to tyrosine yet lacks its redox activity.

The mutated pilin genes were expressed in trans in a PilA⁻ mutantbackground using the expression vector pRG5 (Coppi et al., Appl.Environ. Microbiol. 67: 3180-87 (2001)); Leang et al., BMC Genomics 10,331 (2009). This complementation produced strains that expressed piliwith the Y₅₇F substitution.

Using the same approach, the negatively charged amino acids in thetyrosines' vicinities (D₅₃, D₅₄, and E₆₀) were replaced with anon-charged amino acid, alanine (A), which does not affect the structureof the pilin. Single, double and triple mutations were made. Thus,mutant nanowires with the single E₆₀A replacement, double D₅₃A D₅₄Areplacement, and triple E₆₀A D₅₃A D₅₄A replacement were generated. Amutant strain of G. sulfurreducens that fails to produce pili nanowires(PilA-) was used as a negative control.

More specifically, mutagenesis was performed using the StratageneQuikChange mutagenesis kit. This mutagenesis tool uses pfu Turbo as apolymerase to replicate template DNA from complementary primerscontaining mutagenic nucleotides. The mutagenic oligonucleotides usedfor site-directed mutagenesis are shown below in Table 3.

TABLE 3 Oligonucleotides for Mutagenesis Nucleotide Name replacementSEQ ID Oligonucleotide sequence position(s) Y₅₇F 5′C GCA TTT GCT GAT GAT CAA 200 NO: 12 ACC T T T CCG CCC GAA AG 3′ Y₃₂F 5′CGT GTC AAG GCG T T C AAC 115 NO: 13 AGC GCG GCG 3′ Y₂₇F 5′CCG CAG TTC TCG GCG T T T 100 NO: 14 CGT GTC AAG GC 3′ E₆₀A 5′GAT GAT CAA ACC TAT CCG 209 NO: 15 CCC G C A AGT TAA 3′ D_(53,54)A 5′GAG TCC GCA TTT GCT G C T 188, 191 NO: 16 G C T CAA ACC TAT CCG CCC 3′D_(53,54)A 5′ GAG TCC GCA TTT GCT  188, 191, E₆₀A G C T G CT CAA ACC TAT CCG  209 NO: 17 CCC G C A AGT TAA 3′ S₆₁A 5′GAT GAT CAA ACC TAT 211, 212 NO: 18 CCG CCC GAA G C T TAA 3′The name of the mutated nanowire is provided in the first (left) column,where the nanowire name is the original amino acid one-letter symbolfollowed by the position of the amino acid (as a subscript), which isthen followed by the one-letter symbol for the replacement amino acid.The middle column shows the oligonucleotide sequence with themutagenized codon (in bold) and mutated nucleotide(s) (underlined). Thepositions of the replaced nucleotides in the pilA nucleotide sequenceare shown in the last (right).

The amino acid sequences of the nanowire polypeptides encoded by thesenucleic acids are as follows.

The Y₅₇F polypeptide is shown below as SEQ ID NO: 19.

 1 FTLIELLIVV AIIGILAAIA IPQFSAYRVK AYNSAASSDL 41 RNLKTALESA FADDQT FPPE S

The Y₃₂F polypeptide is shown below as SEQ ID NO:20.

 1 FTLIELLIVV AIIGILAAIA IPQFSAYRVK A F NSAASSDL 41RNLKTALESA FADDQTYPPE S

The Y₂₇F polypeptide is shown below as SEQ ID NO:21.

 1 FTLIELLIVV AIIGILAAIA IPQFSA F RVK AYNSAASSDL 41RNLKTALESA FADDQTYPPE S

The E₆₀A polypeptide is shown below as SEQ ID NO:22.

 1 FTLIELLIVV AIIGILAAIA IPQFSAYRVK AYNSAASSDL 41 RNLKTALESA FADDQTYPP A S

The D_(53,54)A polypeptide is shown below as SEQ ID NO:23.

 1  FTLIELLIVV AIIGILAAIA IPQFSAYRVK AYNSAASSDL 41 RNLKTALESA FA AAQTYPPE S

The D_(53,54)A, E₆₀A polypeptide is shown below as SEQ ID NO:24.

 1  FTLIELLIVV AIIGILAAIA IPQFSAYRVK AYNSAASSDL 41 RNLKTALESA FA AAQTYPP A  S

The S₆₁A polypeptide is shown below as SEQ ID NO:25.

1  FTLIELLIVV AIIGILAAIA IPQFSAYRVK AYNSAASSDL 41 RNLKTALESA FADDQTYPPE A

The constructs were transfected into Geobacter sulfurreducens strainPCA. Cells were routinely grown at 30° C. in NB medium (Coppi et al.,APPL. ENVIRON. MICROBIOL. 67: 3180-87 (2001)) supplemented with 15 mMacetate and 40 mM fumarate (NBAF) before been transferred three times ina modified fresh water (FW) medium (Lovley & Philips, APPL. ENVIRON.MICROBIOL. 54: 1472-80 (1988)), supplemented with 15 mM acetate and 40mM fumarate (FWAF). Briefly, a concentrated (10×) basal FW medium stockcontaining NaHCO₃ (25 g/L), NaH₂PO₄.H₂O (0.6 g/L), NH₄Cl (2.5 g/L), andKCl (1.0 g/L) was prepared. The electron donor and acceptor wereprepared as sterile concentrated stocks (0.75 M sodium acetate and 1 Msodium fumarate, respectively) and the pH of the stock solutions wasadjusted to 7 prior to autoclaving. Vitamins were prepared as separatesolutions as described by Balch et al. (MICROBIOL. REV. 43: 260-96(1979)). Trace minerals were prepared as described by Lovley et al.(APPL. ENVIRON. MICROBIOL. 48: 81-87 (1984)), except that ZnSO₄ wasreplaced with ZnCl₂ (0.13 g/L), and Na₂WO₄.2H₂O (0.025 g/L) was added.FWAF medium contained FW stock (96 ml/L), 0.75 M sodium acetate (20ml/L), 1 M sodium fumarate (40 ml/L), vitamin solution (10 ml/L),mineral solution (10 ml/L) and ddH2O to a final volume of 1 L. Themedium was dispensed in pressure tubes or serum bottles, sparged withN₂:CO₂ (80:20) to remove dissolved oxygen and sealed with butyl rubberstoppers and aluminum tear off seals (Wheaton) prior to autoclaving. Forpili induction, exponentially-growing cells from FWAF cultures grown at30° C. were subcultured in 100 ml of fresh FWAF with 30 mM acetate and40 mM fumarate and the cells were incubated at 25° C. until they reachedearly stationary phase (ca. 72 h).

The conductive properties of the mutant nanowires were measured bytesting the mutant cells compared to wild type and PilA⁻ cells inmicrobial fuel cell assays. Two measurements were made: (i) coulombicefficiency, which measures the amount of electron donor converted intocurrent by the cells, and (ii) the coulombic rates, which measure thecoulombic efficiency per day and are proportional to the electrontransfer rates along the nanowires of the biofilms formed on the anodeelectrode.

Because the nanowires are the electrical connections of the cells in theanode biofilm, defects in their conductivity translated into defects inthe measured coulombic rates. As shown in FIG. 3A, the coulombicefficiency was the same in all the strains, meaning that all the cellsconverted the same amount of electron donor, acetate, into electricitythus ruling out any metabolic defects of the mutations. However, theamino acid replacements resulted in defects in the coulombic rates (FIG.3B). The Y₅₇F substitution produced nanowires with rates of electrontransfer close to a mutant that did not produce the nanowires (PilA⁻),suggesting that the interruption of the electron pathway along thenanowire through the removal of one of the “stepping stones” (atyrosine) produced nanowires with increased resistance to the passage ofelectrons. The replacement of a single negatively-charged amino acid(E₆₀A), which serves as a proton acceptor during electron hopping viatyrosines, resulted in a 1.7-fold decrease in the electron transferrates. Double (D₅₃A D₅₄A) and triple (E₆₀A D₅₃A D₅₄A) mutants producedpili but had coulombic rates comparable to a pilus-deficient mutant.

Another measure of the nanowire conductivity is its ability to reduceinsoluble Fe(III) oxides into soluble Fe(II), which can be measured toindirectly determine the rates of Fe(III) oxide reduction. As shown inFIG. 4, the replacement of negatively charged amino acids also gave riseto a defect in the reduction of Fe(III) oxides.

These results demonstrate that amino acids in the pilin nanowire subunitcan be selectively replaced to modulate the conductive properties of thenanowires pili.

Example 4 Genetic Manipulation of Attachment Points in Pilin Nanowire

This Example illustrates that manipulation of non-redox-active aminoacids can modulate other functions of the nanowires.

For example, some amino acids in the pilin subunit are naturallypost-translationally phosphorylated or modified with glycans. Pilinglycosylation is thought to modulate the binding of pili to varioussurfaces and other cells. Thus, the nanowire's post-translationalmodifications may participate in binding and optimal positioning of theelectron acceptor for electronic coupling.

As illustrated below, genetic engineering of these post-translationallymodified amino acids can be used to manipulate the adhesive propertiesof the nanowires for controlled deposition and efficient electroniccoupling in integrated nanocircuits and other nanodevices. Thepost-translational modifications also affect the nanowire's charge and,therefore, could contribute to its conductive properties.Phosphorylation can affect the pilus charge, which could affect chargetransport and the binding properties of the nanowires. Glycansencapsulating metallic nanowires reduce atomic contacts with the aqueousenvironment and minimize electronic fluctuations (see, Leroux et al., J.Am. Chem. Soc. 130: 13465-70 (2008)).

Post-translational modification of the nanowire subunit can bemanipulated via genetic engineering to modulate the efficiency of thenanowire's conductive properties. A serine residue in C-terminalposition 61 (S₆₁) may be glycosylated. Site-directed mutagenesis wasused to replace this C-terminal serine (S₆₁) with an alanine, andgenerate cells that express a mutant nanowire S₆₁A.

As shown in FIG. 5A, the S₆₁A nanowires had a defect in Fe(III) oxidereduction that may be caused by defective binding and/or defectiveconductivity of the nanowires. Thus, the conductive properties of theS₆₁A nanowires were tested in microbial fuel cell assays. In this case,the coulombic efficiencies and coulombic rates were the same in thewild-type and S₆₁A nanowires, demonstrating that the conductiveproperties of the nanowires were unaltered.

Thus, the defect in Fe(III) oxide reduction but the existence ofwild-type coulombic rates in the S₆₁A nanowires indicates that thereplacement of serine at position 61 affected the adhesive properties ofthe nanowires. Thus, genetic engineering can be used to manipulateproperties of the nanowires other than conductivity to suit specificapplications such as the controlled deposition of the nanowires onvarious surfaces.

Example 5 Method I for Purifying Pili to Homogeneity

In experiments performed by the inventors it was observed that the piliof G. sulfurreducens did not depolymerize using mild denaturationmethods, including standard conditions with sodium dodecyl sulfate (SDS)detergent and heat treatment routinely used for denaturingSDS-electrophoresis (Laemmli, Nature 227, 680-685 (1970)). The observedbiochemical resistance was due to the intrinsic resistance of thenanowire filaments to depolymerize in the presence of detergents, aswell as their tendency to aggregate and form thick bundles. These thickbundles were also observed to be more resistant to depolymerization anddenaturation than the individual pilus. This property suggests that thepili of G. sulfurreducens are very stable protein assemblies. As aresult, selective separation of pili from other proteins, viapreparative denaturing SDS electrophoresis, was chosen as a purificationprocedure (see, e.g., Collinson et al., J. Bacteriol. 173, 4773-4781(1991)).

Geobacter Source

The bacterium Geobacter sulfurreducens strain PCA (Gsu) was obtainedfrom the American Type Culture Collection (ATCC) where it is registeredunder accession number ATCC® 51573™. It was obtained as a substantiallypure culture and maintained under conditions typically used in the artwithin the inventors' laboratory culture collection. All chemicals,including vitamins, were from Sigma-Aldrich and had a minimum purity of98%.

Bacterial Growth Conditions

The Gsu PCA strain was used throughout the study. Cells were routinelygrown at 30° C. in NB medium supplemented with 15 mM acetate and 40 mMfumarate (NBAF) before being transferred three times to a modified freshwater (FW) medium supplemented with 15 mM acetate and 40 mM fumarate(FWAF) (see, e.g., Coppi et al. Appl. Environ. Microbiol. 67, 3180-3187(2001); Lovley & Phillips, Appl. Environ. Microbiol. 54, 1472-1480(1988)). A concentrated (10×) basal FW medium stock containing NaHCO₃(25 g/L), NaH₂PO₄.H₂O (0.6 g/L), NH₄C (2.5 g/L), and KCl (1 g/L) wasprepared. The electron donor and acceptor were prepared as sterileconcentrated stocks (0.75 M sodium acetate and 1 M sodium fumarate,respectively) and the pH of the stock solutions was adjusted to 7 priorto autoclaving.

Vitamins were prepared as separate solutions as previously described byBalch et al. (Microbiol. Rev. 43, 260-296 (1979)). Trace minerals wereprepared as described by Lovley et al. (Appl. Environ. Microbiol. 48,81-87 (1984)), except that ZnSO₄ was replaced with ZnCl₂ (0.13 g/L), andNa₂WO₄.2H₂O (0.025 g/L) was added. FWAF medium contained FW stock (96mL/L), 0.75 M sodium acetate (20 ml/L), 1 M sodium fumarate (40 ml/L),vitamin solution (10 mL/L), mineral solution (10 ml/L) and ddH₂O to afinal volume of 1 L. The medium was dispensed in pressure tubes or serumbottles, sparged with N₂:CO₂ (80:20) to remove dissolved oxygen andsealed with butyl rubber stoppers and aluminum tear off seals (Wheaton)prior to autoclaving. For pili induction, exponentially-growing cellsfrom FWAF cultures grown at 30° C. were subcultured in 100 ml of freshFWAF with 30 mM acetate and 40 mM fumarate and incubated at 25° C.(Reguera et al. Nature 435, 1098-1101 (2005)) until they reached earlystationary phase (ca. 72 h).

Pili Isolation and Purification

Pili were purified to homogeneity using a modification of the protocolby Collinson et al. (J. Bacteriol. 173, 4773-4781 (1991)).Pili-expressing cells were harvested by centrifugation (13,000×g) for 15min at 25° C. and resuspended in 6 ml of 10 mM Tris HCl, pH 8.0 (Trisbuffer; Invitrogen, 99.9%). The cells were lysed by sonication(five×1-min at 4° C. per 1 ml of cell suspension; Branson Sonifier 450)before adding RNase A (bovine pancreas; Roche Diagnostics) and DNase I(bovine pancreas, Sigma, 91% purity) enzymes to a 0.1 mg/ml finalconcentration and MgCl₂ (J. T. Baker, 99.4%) to a final concentration of1 mM. The cell extracts were incubated at 37° C. for 20 min toenzymatically degrade the nucleic acids in the sample. Lysozyme (hen eggwhite, Roche Diagnostics) was then added to a concentration of 1 mg/mland incubated at 37° C. for 40 min with gentle shaking (200 rpm, Innova4340, New Brunswick). Cell membranes and proteins in the extract weresolubilized with sodium dodecyl sulfate (SDS, 1% final concentration;Sigma 98.5%) after incubation at 37° C. for 30 min. The SDS-insolublefraction was collected by centrifugation (12,100×g, 15 min, 25° C.) andwashed twice with 6 ml of Tris buffer. The SDS-insoluble fraction inTris buffer was digested again with RNase, DNase and lysozyme, asdescribed above. Mechanical vortexing (Fisher Scientific) was used tomix the samples. Samples with large clumps and/or aggregates weresubjected to 2 additional cycles of sonication for 1 min at 4° C. Theinsoluble fraction was collected as described above, washed twice, andresuspended in 1 ml of Tris buffer. When needed, the sample was storedat −20° C. overnight.

The protein sample was suspended in 2 ml of SDS-polyacrylamide gelelectrophoresis (PAGE) sample buffer (10% [v/v]glycerol; 5%[v/v]b-mercaptoethanol; 2% [w/v] SDS, and 62.5 mM Tris HCl, pH 6.8) andboiled for 15 min. The SDS-treated sample was loaded on top of apreparative 12% polyacrylamide gel with a 5% stacking gel, and subjectedto electrophoresis at 40 mA for 5 h using Prep Cell 491 (Bio-Rad). Thematerial that did not enter the stacking gel was recovered by aspirationwith a pipette and washed three times with 1 ml ddH₂O of doubledistilled water by centrifugation (12,100×g, 15 min, 25° C.). Theprotein in the SDS-insoluble fraction was extracted twice with 95%ethanol (Decon Laboratories) and lyophilized or dried in a Speed Vacsystem (Savant Instruments Inc) at room temperature for approximately 20min. This ethanol step also solubilized organic contaminants such asquinone-like compounds or organic cofactors. The dried protein wasresuspended in 1 ml of ddH₂O and vortexed for 60 seconds to break up thelarge clumps. Poorly-bound protein in the insoluble material wasextracted with 0.2 M glycine (pH 1.5, adjusted with HCl; Invitrogen) at100° C. for 10 min. The insoluble fraction was recovered bycentrifugation (16,000×g, 25 min, 4° C.), washed five times with ddH₂O,and lyophilized or dried in a SpeedVac at room temperature untilcompletely dry. The dried sample was then stored at −20° C. forshort-term use or flash frozen in liquid nitrogen for long-term use.

Quantitative elemental analysis of the purified pili preparations wasperformed by Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES) using a Thermo Jarrell-Ash Enviro 36 Inductively Coupled ArgonPlasm (Chemical Analysis Laboratory, University of Georgia, Athens). Forthese experiments, 1 ml aqueous samples of purified pili containing40-70 micrograms of protein per milliliter were analyzed in reference toblank control samples (without protein). When indicated,ethylenediaminetetraacetic acid (EDTA, Invitrogen) was added to thesample at a final concentration of 0.1 mM prior to the ICP-AES analyses.Protein concentration was determined using the bichinchoninic acid (BCA)assay (Smith et al., (Anal. Biochem. 150, 76-85 (1985); Pierce®, ThermoScientific) with Bovine Serum Albumin (BSA) as the protein standard.

Protein Electrophoresis

Dried pili preparations were resuspended in 15 ml of ddH₂O containing 2%(w/v) Octyl-β-D-Glucopyranoside (OG) (Sigma, 98%) and incubated at roomtemperature for 1 h prior to SDS-PAGE. SDS-PAGE was performed accordingto the method of Laemmli (Nature 227, 680-685 (1970)) as modified byAmes (J. Biol. Chem. 249, 634-644 (1974)). The OG-treated sample wasboiled in SDS-PAGE sample buffer (Laemmli, Nature 227, 680-685 (1970))and subjected to electrophoresis on 12% ReadyGels (Bio-Rad) using a MiniProtean Tetra Cell apparatus (Bio-Rad). After electrophoresis, the gelswere silver stained using the Pierce® Silver Stain for Mass Spectrometrykit (Thermo Scientific), following the instructions supplied bymanufacturer.

After silver-staining, the ca. 7-kDa PilA protein band was excised fromthe gel, destained and digested with trypsin following the proceduredescribed by manufacturer (Pierce® Silver Stain for Mass Spectrometry,Thermo Scientific). The peptides in the tryptic digest were concentratedand purified chromatographically with C₁₈ reversed-phase media (ZipTip®,Millipore) and separated by matrix assisted laser desorptionionization-time of flight spectrometry (MALDI-TOF, Shimadzu Axima).Peptide identification and prediction of potential contributions ofpost-translational modifications to the peptide mass was performed usingthe MS-DIGEST tool at the ProteinProspector database from the Universityof California, San Francisco, see website atprospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msdigest.

Western Blot (Immunoblot) Analysis

Proteins separated by SDS-PAGE were electrophoretically transferred to anitrocellulose membrane (HyBond ECL™, Amersham GE Healthcare) at 50 Vfor 15 min using a Mini Protean Tetra Cell apparatus (Bio-Rad). Therapid western blotting kit (Amresco®) was used for the electrophoretictransfer and membrane blocking, following manufacturer'srecommendations. After blocking, the membrane was incubated in 10 mlrapid antibody diluent solution (45 min, room temperature, gentleagitation) with a 1:5,000 dilution of the primary antibody (rabbitα-PilA polyclonal antibodies raised against the 42 amino acids at thecarboxy-terminus of the PilA protein) and a 1:2,500 dilution of goatα-PilA rabbit IgG antibodies conjugated to the Cy^(tm) 5 fluorescencedye (ECL™ Plex, Amersham GE Healthcare). The membrane was washed inrapid wash solution provided by manufacturer (3 times, for 5 min). Themembrane was then scanned with Typhoon imager operated in fluorescencemode (excitation at 633 nm, 670 BP 30 filter, and PMT setting at 600 V)to visualize the protein bands that hybridized with the primaryantibodies.

Microscopy

For Confocal Laser Scanning Microscopy (CLSM), dried preparations ofpurified pili were dissolved in phosphate buffer saline (PBS), depositedon the surface of a glass cover slip and allowed to adsorb for 30 min.The adsorbed pili were then washed with PBS and fixed with 100microliters of 3.7% paraformaldehyde in PBS. After washing with PBS, thesamples were incubated for 30 min in PBS containing 1% BSA, beforeadding the anti-PilA primary antibody (1:100) and incubating at 4° C.overnight. Following three washes in PBS-1% BSA, the samples wereincubated with the secondary antibody (α-PilA rabbit conjugated to Alexafluor 488 dye, 1:1000) for 1 h. The cover slip was then washed threetimes with PBS buffer and examined with Zeiss LSM Pascal confocalmicroscope equipped with a Plan-Neofluar 63x oil objective (excitation,488 nm; emission, 505-535 nm).

For transmission electron microscopy (TEM), an aqueous solution ofpurified pili was adsorbed on a carbon-copper grid (Mesh 300. ElectronMicroscopy Sciences), negatively stained with 1% (w/v) uranyl acetate indistilled water. The negatively stained samples were examined with aJeol 100 CX electron microscope (Japan Electron Optic Laboratory)operated at 100 kV.

Distal (lateral) and axial (length) conductivity measurements wereperformed, respectively, by scanning tunneling microscopy (STM) andconductive probe-atomic force microscopy (CP-AFM). STM imaging andspectroscopy was performed as described by Veazey et al. (Electronicstructure of Geobacter sulfurreducens pilus nanowires probed by ScanningTunneling Microscopy. Phys. Rev. B (to be published in 2011)). ForCP-AFM measurements a Bio-AFM-CF instrument (Asylum) was used. A goldelectrode grid nanofabricated onto a silicon substrate was used forbiological deposition. For the fabrication of the gold grid, photoresist(Shipley S1813) was spin-coated onto silicon wafers having a 300 nmthermal oxide layer (SiO₂). After photoresist development, patternedgold electrodes were deposited by thermally evaporating 5 nm of titaniumfollowed by 25 nm of gold onto the surface of the wafer. A solutioncontaining ca. 40-70 micrograms of purified pili protein per milliliterin ddH₂O were then deposited onto the electrodes, left to adsorb for 25minutes, and then wicked dry with absorbent paper. CP-AFM was performedwith Pt-coated cantilevers having spring constant 2 N/m (Veeco). Pilusnanowires lying across the gold-SiO₂ interface were first identified inimaging mode. For Current (I) versus voltage (V) (“I-V”) measurements,the tip was placed on a point of the pilus lying on the SiO₂. Positivecontrols were generated by positioning the tip on the gold electrode,while negative controls were produced by positioning the tip on the SiO₂substrate at 100-nm distances from the gold edge.

Results

Transmission electron microscopy (TEM) of negatively-stainedSDS-insoluble samples confirmed the presence of bundles of fibrilsmorphologically similar to the pili displayed on the surface of cells ofG. sulfurreducens. Furthermore, the fibrils were free of obviouscellular debris. The presence of the PilA subunit in the purifiedfibrils was detected immunologically by confocal laser scanningmicroscopy (CLSM) of purified fibrils hybridized to polyclonalantibodies raised against a recombinant truncated form of the PilA pilinsubunit (anti-PilA) and fluorescently-labeled secondary antibodies.Atomic Force Microscopy (AFM) was used to image the purified pili fromG. sulfurreducens that were deposited on a HOPG substrate. The resultingAFM image of the pilus fiber shows that the average width of the fibrilswas in the 4-5 nm range.

Denaturing SDS-PAGE and immunodetection by Western blot using polyclonalanti-PilA antibodies was used to investigate the protein composition ofthe pili and asses its purity. Standard denaturation conditions with SDSdetergent and heat treatment did not fully depolymerize the pili intothe pilin subunit. However, treatment with octyl-glucoside partiallydepolymerized the pili into oligomers of various sizes and a proteinband that migrated as between 3 and 10 kDa in the gel, consistent withthe predicted size of the mature PilA protein (6.5 kDa without anypost-translational modifications). After electrophoresis, a denaturing4-20% SDS-polyacrylamide gel showed pili oligomers in untreated controlsand pilus oligomers of various sizes as well as the ca. 7 kDa PilA bandin samples pretreated with 1% or 2% octyl-glucoside. The 7 kDa band waspositively detected as PilA in immunoblots using anti-PilA antibodies.

The 7-kDa protein band that was electrophoretically separated afterdepolymerizing purified pili with 2% octyl-glucoside was extracted fromthe gel, digested with trypsin and the mass of the tryptic peptides wasanalyzed by MALDI-TOF. Peptide mass fingerprinting of the tryptic digestby MALDI-TOF identified several PilA peptides, some carrying potentialpost-translational modifications, such as the N-methylation of thephenylalanine at the peptides amino-terminus that all pilins have.Details of peak assignment are presented in Table 4. These dataconfirmed the identity of the PilA band and demonstrate that the pili ofG. sulfurreducens were selectively purified to homogeneity.

TABLE 4 Masses of Tryptic Peptides of PilA from Geobacter sulfurreducensDetected with MALDI-TOF Mass Spectrometry Expected Observed SEQ  Modifi-Peak mass mass Sequence^(a) ID NO: cation 1 2205 2207 (K)TALESAFAD 262 phospho, DQTYPPES acetyl 2 2220 2221.6 (K)TALESAFAD 27 2 phospho,DQTYPPES 3 2280 2280.4 (R)NLKTALESA 28 methyl FADDQTYPPES 4 2295 2294.9(R)NLKTALESA 29 2 methyl FADDQTYPPES 5 2309 2308.5 (R)NLKTALESA 30acetyl FADDQTYPPES 6 3326 3323.9 FTLIELLIVVAII 31 — GILAAIAIPQFSAYRVKA(V) 7 3341 3337.6 FTLIELLIVVAII 32 methyl GILAAIAIPQFSA YRVKA(V) 83356 3353.6 FTLIELLIVVAII 33 2 methyl GILAAIAIPQFSA YRVKA(V) ^(a)Anamino acid in parenthesis is a trypsin cleavage position.

The lack of proteins, other than PilA, in the pili fractions excludedthe possibility of c-cytochromes being associated with the pili.However, it did not exclude the possibility of metals being directlybound to the pilus shaft. Amino acid residues can be positioned in thefolded protein to form structural motifs for metal coordination. Boundmetals not only enable electron transfer reactions but also stabilizethe protein's secondary structure (Reguera et al. Nature 435, 1098-1101(2005); Haas & Franz, Chem. Rev. 109, 4921-4960 (2009)).

The SeqCHED server (Levy et al. Proteins 76, 365-374 (2009)) was usedwithin the SPACE tools suite (Sobolev et al. Nucleic Acids Res. 33,W39-43 (2005)) to identify soft (Zn, Fe, Ni, Cu, Co, Mn) andpromiscuous, hard (Mg, Ca) metal-ion binding sites in the mature PilAamino acid sequence. However, none were identified.

Despite the lack of conserved metal-binding sites in the pilin, theassembly of pilin subunits to form the pilus shaft could createstructural and sequence motifs for metal coordination. To investigatethis, Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES)was used for elemental analysis of aqueous samples of the purified piliin reference to protein-free ‘blank’ controls. This technique has beentraditionally used for metal analyses of metalloproteins because of itshigh specificity and sensitivity at the identification andquantification of trace elements based on the distinct energy thatexcited electrons emit at a given wavelength as they return to groundstate (Ma et al. Electrophoresis 25, 2469-2477 (2004)). No significantdifferences were observed between samples and protein-free controls formost of the elements analyzed. These results are shown in Table 5 below.

TABLE 5 Elemental analyses by ICP-AES of purified pili in the absence orpresence of EDTA Lower LOD Lower LOD Elements Elements/EDTA Metal(ppm)^(a) (10¹⁶ atoms)^(b) (10¹⁶ atoms)^(c) (10¹⁶ atoms)^(d) Al 0.060.13 0.464 ± 0.349 <LOD Sb 0.09 0.05 As 0.08 0.06 Ba 0.06 0.03 <LOD <LODBe 0.09 0.60 B 0.10 0.56 <LOD <LOD Cd 0.06 0.03 <LOD <LOD Ca 0.05 0.080.222 ± 0.192 <LOD Cr 0.06 0.07 <LOD <LOD Co 0.06 0.06 <LOD <LOD Cu 0.070.07 <LOD Fe 0.05 0.05 0.118 ± 0.1  <LOD Pb 0.06 0.02 <LOD <LOD Mg 0.030.07 0.134 ± 0.116 <LOD Mn 0.10 0.11 <LOD <LOD Mo 0.05 0.03 <LOD <LOD Ni0.10 0.10 <LOD <LOD P 0.09 0.18 4.499 ± 3.906 <LOD K 0.50 0.77 3.671 ±3.120 <LOD Se 0.09 0.07 Si 0.50 1.07 <LOD <LOD Ag 0.10 0.06 Na 0.50 1.3138.337± 6.867 Sr 0.05 0.03 <LOD <LOD Tl 0.05 0.01 Ti 0.10 0.13 V 0.150.18 Zn 0.05 0.05 <LOD <LOD ^(a)Instrumental Limits of Detection (LOD)^(b)Calculated for total volume of 1 ml. ^(c)Elements detected inpurified pili preparations (<LOD, lower than lowest detection limits)^(d)Elements detected in purified pili preparations after EDTA treatment(<LOD, lower than lowest detection limits)

The pili were also treated with low (0.1 mM) concentrations of EDTA toremove weakly bound elements carried over during the course ofpurification. Among the metal ion cofactors known to catalyze electrontransfer (Fe, Cu, Mo) or redox (Fe, Cu, Mn, Co and Ni) reactions, onlyFe was detected (0.19±0.14 atoms per pilin). However, as indicated bythe data in Table 5, Fe levels varied widely from sample to sample,suggesting it was a trace contaminant rather than a tightly boundcofactor. Mild treatment with EDTA effectively removed it from the pilisamples.

Quinones such as ubiquinones and menaquinones are lipid solublemolecules that function as the primary electron carriers of thebacterial inner membrane and serve as electronic link to membrane-boundrespiratory complexes, a process that requires quinones to bind tospecific structural motifs in quinone-reactive redox proteins (Fisher &Rich, J. Mol. Biol. 296, 1153-1162 (2000); Gunner et al. J. Bioenerg.Biomembr. 40, 509-519 (2008); Simon & Kern Biochem. Soc. Trans. 36,1011-1016 (2008)). Because of their hydrophobic nature, quinones bindmotifs located in hydrophobic regions of redox proteins (Fisher & Rich,J. Mol. Biol. 296, 1153-1162 (2000)). Type IV pili are predicted to havea narrow (6-11 Å) hydrophobic central channel. Because the pilus isanchored on the inner membrane of Gram-negative bacteria, its innerhydrophobic channel could potentially house quinones and create aninternal pathway for electron transfer free of solvents. However,fluorescence spectroscopy of the purified nanowires revealed no emissionpeak in the 400-500 nm emission ranges of all the known quinones (SeeCory and McKnight. Fluorescence Spectroscopy Reveals Ubiquitous Presenceof Oxidized and Reduced Quinones in Dissolved Organic Matter. Environ.Sci. Technol. 2005, 39, 8142-8149). An emission peak at a 305 nmwavelength was detected, but this peak corresponds with nanowiretyrosine residues and not to quinones.

Moreover, when the nanowire structure was opened up using partialdenaturation of the nanowire with 8M urea for two (2) hrs at roomtemperature, no internal quinones were exposed as a result. Denaturedsamples showed the tyrosine peak at above 300 nm and a large peak closeto 350 nm, which is consistent with loss of tyrosine fluorescencequenching as the protein structure is denatured.

The results presented thus far demonstrated the proteinaceouscomposition of the purified pili, and rule out its association with bothorganic and inorganic redox-active cofactors.

The purified pili were also used to investigate the contribution of thepilus protein matrix to extracellular electron transfer in G.sulfurreducens. Scanning tunneling microscopy (STM) was used to probethe axial (lateral) conductivity of purified pili. STM provides a higherspatial resolution probe and permits more direct electroniccharacterization compared to the CP-AFM approach used in earlier work todemonstrate the conductive nature of mechanically-sheared pilipreparations. No chemical fixation was used to prevent potentialartifacts. Applying a voltage causes electrons to tunnel from occupiedstates at the sample surface into unoccupied states of the tip, or viceversa. As the amount of tunneling current is proportional to the numberof available electronic states, STM can probe the local density ofstates of the pili and measure the contribution of individual aminoacids in the protein matrix. The method has been successfully applied tostudy the surface topography and electronic properties of biologicalsamples.

STM imaging of purified pili showed periodic conducting filaments of theexpected diameter (FIGS. 6A and 6B). Bright spots were observed in thepilus. These bright spots are not taller in the topographical sense, butrepresent regions of the pilus that supply more tunneling current due toan increase in the local density of states. These bright locations areprobably due to molecular sub-structures, consistent with the presenceof conductivity ‘hot spots’ such as redox-active amino acids. Current(I) versus voltage (V) measurements taken at various locations of thepilus confirmed the metallic (ohmic) behavior at biological (±1 V)voltages (FIGS. 6A and 6B).

Controls with non-conductive purified pili from Pseudomonas aeruginosastrain K (PAK) were used to demonstrate the insulating behavior of otherpili at biologically-relevant voltages. Large sample voltages, greaterthan 2 V, were necessary to image the PAK pili. At these voltages, tipinstabilities often result due to the large electric field between thetip and the sample, which causes distortions and noise in the imageddata. This was even more pronounced for the pilus nanowires, due to thehigh tunneling rates produced by such highly conductive materials athigh-voltages. The PAK pili thus serve as insulating controls in the ±1V range, which represent biologically relevant voltages (often in the mVrange). The PAK's distal conductivity at high voltages possibly reflectselectron transfer through the peptide backbone. These resultsdemonstrate that the protein casing of the pilus nanowires can conductelectrons laterally at ±1 V voltage ranges. In contrast, structurallysimilar protein filaments such as the PAK pili cannot. These resultssuggest that specific amino acids in the pilus nanowires function aselectronic conduits to promote electron transfer reactions.

The STM studies presented above probed the distal conductivity of thepilus, thereby demonstrating that the protein matrix conducted electronsat distances in the same ranges as the pilus diameter (4-5 nm). In orderto establish the upper range that the pilus protein matrix can conductelectrons, CP-AFM was employed to probe the axial (length) conductivityof pili deposited onto nanofabricated gold grids lying on an insulatingsilicon substrate. The conductive AFM tip was used to probe theconductivity of the pilus on the gold electrode (equivalent to thedistal measurement) and these measurements were compared to the currentmeasured when the tip was positioned at various points on the pilusfilament lying on the insulating silicon substrate.

FIG. 6C demonstrates that Geobacter pilli can transfer electrons alongtheir length. The data used for generating FIG. 6C were raw(non-normalized) data. However, curve 702 shows the current versusvoltage with the AFM tip touching the purified pilus. As curve 702shows, there is electrical conductivity along the axial length of thenanowire. The same pilus touches a gold surface electrode 200 nm awayfrom the AFM tip. Therefore, the measurement shows the current passingfrom the tip, through the nanowire, and to the gold electrode. Curve 704indicates that the AFM tip was touching the Si substrate 100 nm awayfrom the gold edge and was not touching a nanowire. No measurablecurrent results were observed.

For the positive control 706, the AFM tip was directly positioned on thegold electrode. As FIG. 6C shows, the conductivity of the directtip-to-gold pathway was large.

The I-V curves clearly demonstrate, for the first time, the conductingbehavior of the pilus with respect to axial conductivity, consistentwith the conducting behavior previously demonstrated in distalmeasurements by CP-AFM (Reguera et al. Nature 435, 1098-1101 (2005)).Moreover, this behavior is consistent with similar mechanisms of theprotein matrix mediating both distal and axial charge transport in thepili.

These results demonstrate that the protein matrix of the pili of G.sulfurreducens can, by itself, catalyze electron transfer reactions atdistances that greatly exceed the known limits for protein-basedelectron transfer.

Furthermore, these results rule out any contribution from organic andinorganic cofactors known to mediate long-range electron transfer inproteins and support, instead, a mechanism exclusively mediated by theprotein matrix. A solvent-free pathway through the pilus central channelmediated by quinones is not plausible because external organic moleculeswere extracted with ethanol from the conductive pili during purificationand no emission peaks were detected by fluorescence spectroscopy topurified nanowires or partially denatured nanowires. The hydrophobicnature of the inner pilus channel also prevents solvents from fillingthe pilus internal cavity, so that electron transfer can proceedefficiently through an inner electrolytic channel. In support of this,elemental analyses by ICP-AES detected only trace amounts of the alkalimetal sodium after removing weakly bound ions with EDTA. See Table 6below.

TABLE 6 Atoms per pilin detected by ICP-AES analysis of pili samplesatoms/pilin Std Dev^(a) Al 0.801 0.252 Ca 0.348 0.301 Fe 0.189 0.140 K5.835 4.561 Mg 0.209 0.182 Na 60.080 52.080 P 7.050 6.121 ^(a)Standarddeviation of three replicates

The measured concentrations varied from sample to sample withoutaffecting the conductivity measurements of the pili preparations,suggesting they do not provide sufficient ionic strength to contributeto the pilus conductance. The pilus nanowires also lacked associatedmetals that are biologically relevant. From all the inorganic elementsknown to participate in electron transfer and redox catalysis inbiological systems, only some low levels of iron were detected insamples that were not treated with EDTA. Again, there was a lot ofvariability from sample to sample, with samples lacking any detectablelevels of Fe, yet producing consistent I-V curves by STM and CP-AFM.Samples with the highest levels of Fe had the equivalent of one atom ofiron per 5 pilin subunits assembled along the pilus shaft.

With an estimated assembly of 3.6 pilin subunits per turn and a piluspitch of 37 Å, the distance between potential iron redox centers in thepilus would exceed 51 Å. This is close to 5 times the optimum distance(<14 Å) established for electron transfer between metal-containing redoxcenters. Other metals such as boron or cadmium, which are commonly usedto metalize insulating materials during the manufacturing of inorganicnanowires were also below the limits of detection. Electron transferover distances beyond the 25 Å theoretical and experimental tunnelinglimits supports the involvement of multistep tunneling (hopping),single-step superexchange tunneling pathways and/or yet to be discoveredtransport mechanisms mediated by the protein matrix.

Example 6 Method II for Purifying Pili to Homogeneity

In this example, pili, purified as assemblies of a single peptidesubunit, the PilA pilin, and without any associated proteins, such asc-cytochromes, or metals are shown to be conductive. Metals, ions andother known redox cofactors such as flavins and quinones were alsoabsent.

Starting Materials

The starting materials as well as the bacterial strains and cultureconditions were as described in Example 5.

Isolation and Purification of Pili

Pili were purified to homogeneity as described in Example 5 except thatall the buffers used during the purification contained 1 mMethylenediaminetetraacetic acid (EDTA) and all drying steps were carriedout with a constant flow of filter-sterilized N₂ gas rather than in aSpeed Vac (which may introduce contaminants in the pili samples).

Unless otherwise indicated, dried preparations of purified pili wereresuspended in 10 mM CHES buffer containing 1 mM EDTA and incubated fora minimum of 24 h at 4° C. to deaggregate the pili bundles that formedduring purification. A 2:1 (v/v) chloroform-methanol solution was thenadded to the pili samples to extract quinone-like contaminants followingthe method in F. Brito, J. A. DeMoss, M. Dubourdieu, J. Bacteriol. 177,3728 (July, 1995). After 2 h at 4° C., the chloroform phase was removedand discarded. The methanol was then evaporated with a constant flow offilter-sterilized N₂ gas and the dry pili sample was stored at −20° C.for short-term use or flash frozen in liquid nitrogen and stored at −80°C. for long-term use.

Pseudomonas aeruginosa strain K (PAK) pili were purified as described inW. Paranchych et al., Can. J. Microbiol. 25, 1175 (October, 1979), withsome modifications. Briefly, PAK cultures grown in TBS tolate-exponential phase were plated on TBS agar plates and grownovernight at 37° C. The cells were harvested from the plates andsuspended in standard saline citrate buffer, SSC (1 g of wet weight per10 ml SSC). Pili and flagella were sheared off the cells mechanically bystirring the cell suspension at 4° C. for 2 h and vortexing 5 times (1min each cycle). Bacterial cells were removed by centrifugation (8000×g,20 min). Pili and flagella were precipitated out of the supernatantfractions with NaCl (0.5 M) and polyethylene glycol 6000 (PEG 6000, 1%w/v) after overnight incubation at 4° C. The pili and flagellaprecipitates were harvested by centrifugation (6000×g, 25 min) andseparated after incubating the samples at 4° C. in a 10% w/v (NH₄)₂SO₄solution (pH 4.0) for 2 h. After centrifugation (6000×g for 15 min), thesupernatant fraction containing the flagella was discarded. Threesequential steps of ammonium sulfate precipitation were used to removeany remaining flagella from the pili samples. The final pellet,containing the purified PAK pili, was resuspended in ddH₂O and dialyzedfor 24 h to remove any remaining (NH₄)₂SO₄. The dialyzed solution wasused for STM experiments.

Protein concentration in the pili preparations was determined with thebichinchoninic acid (BCA) assay (S8) (Pierce®, Thermo Scientific) andusing 60° C. incubations for 1 h. Bovine Serum Albumin (BSA) was used asthe protein standard.

Microscopy

For Transmission Electron Microscopy (TEM), purified pili wereresuspended in ddH₂O (adjusted to pH 7) to visualize the pili bundles orin 10 mM CHES buffer (pH 9.5) and incubated at room temperature for 72 hto promote deaggregation. These samples were adsorbed onto a 300-meshcarbon-copper grids (Electron Microscopy Sciences), negatively stainedwith 1% (w/v) uranyl acetate, and allowed to dry, as described inExample 5. The samples were examined with a Jeol 100 CX electronmicroscope (Japan Electron Optic Laboratory) operated at 100 kV.

For Scanning Probe Microscopy, pili samples were routinely deposited onfreshly cleaved highly oriented pyrolytic graphite (HOPG) and imagedwith an atomic force microscope (AFM), as described in Example 5. Distal(lateral) and axial (length) conductivity measurements were performed byscanning tunneling microscopy (STM) and conductive probe-AFM (CP-AFM),respectively. For STM, dried preparations of G. sulfurreducens or PAKpili were resuspended in phosphate buffer saline (PBS) and deposited for15-30 min. The excess liquid was wicked with absorbent paper and theHOPG surface was dry under a stream of N₂ gas. Applying a voltage withthe STM causes electrons to tunnel from occupied states at the samplesurface into unoccupied states of the tip, or vice versa. As the amountof tunneling current is proportional to the number of availableelectronic states. STM was used to probe the local density of states ofthe pili. This technique also provides a higher spatial resolution probeand more direct electronic characterization compared to conventionalconducting probe-atomic force microscopy (CP-AFM) approaches.

For Scanning Tunneling Microscopy (STM) images were acquired at constantsample voltages, as indicated, by scanning while keeping the tunnelingcurrent constant with the use of a feedback circuit. The apparent STMwidth of the pilus fibers was obtained from cross sections and thebroadening effect was corrected as described in Biró, L. P et al.Scanning tunneling microscopy (STM) imaging of carbon nanotubes. Carbon36:689-696 (1998). The axial electronic structure of the pilus fiberimaged by STM was also generated to identify electronic(voltage-dependent) and topographic (present at all voltages)periodicities. I-V curves were obtained with the tip positioned on thecenter of the pilus filaments while suspending the feedback and rampingthe bias voltage.

The tunneling conductance, dI/dV, was calculated as the numericaldifferentiation of the I over V values and plotted against thetip-sample bias (voltage. V) to investigate the density of states of theG. sulfurreducens pili as a function of energy and in reference to thePAK pili controls.

Conducting probe-atomic force microscopy (CP-AFM) was used to probe theaxial conductivity of the G. sulfurreducens pili with a Bio-AFM-CFinstrument (Asylum). The substrate used for pilus deposition andmeasurements was a gold electrode grid nanofabricated onto a siliconsubstrate. For the fabrication of the gold grid, photoresist (ShipleyS1813) was spin-coated onto silicon wafers having a 300 nm thermal oxidelayer (SiO₂).

After photoresist development, patterned gold electrodes were depositedby thermally evaporating 5 nm of titanium followed by 25 nm of gold ontothe surface of the wafer. A solution containing ca. 5 μg/ml of purifiedpili in ddH₂O with 1 mM EDTA was then deposited onto the electrodes,left to adsorb for 10-20 min, and then wicked dry with absorbent paper.CP-AFM was performed with Pt-coated cantilevers having spring constant 2N/m (Veeco).

Pilus nanowires lying across the gold-SiO₂ interface were firstidentified in imaging mode. For I-V measurements, the tip was placed ona point of the pilus lying on the SiO₂. Positive controls were generatedby positioning the tip on the gold electrode, while negative controlswere produced by positioning the tip on the SiO₂ substrate at 100-nmdistances from the gold edge. The resistance of the pilus was calculatedfrom the slope of the linear current-voltage (I-V) plot. Ohm's law(I=V/R) was used to estimate the current (in Amps) along the pilus. If apotential of 100 mV were applied across the length of this pilus, thecurrent would be I=V/R=1e-1/2e8=5e-9=5 nano amps (=5 nA).

Protein Electrophoresis and Immunoblot Analyses

Dried preparations of purified pili were resuspended in 5 ml of ddH₂Ocontaining 10% (w/v) Octyl-b-D-Glucopyranoside (OG) (Sigma) andincubated at room temperature for 2 h. The concentration of OG wasadjusted to 2% (v/v) and the solution was incubated for an additional 24h at room temperature prior to SDS-PAGE. The OG-treated sample wasboiled in SDS-PAGE sample buffer (S9) and subjected to electrophoresison 10-20% Tris/Tricine ReadyGels (Bio-Rad) using a Mini Protean TetraCell apparatus (Bio-Rad). After SDS-PAGE separation, the proteins in thegel were electrophoretically transferred to a PVDF membrane (HyBondLFP™, Amersham GE Healthcare) at 25 V for 150 min using a Mini ProteanTetra Cell apparatus (Bio-Rad).

The Amersham ECL Plex Western blotting kit was used for theelectrophoretic transfer and membrane blocking, following manufacturer'srecommendations. After blocking, the membrane was incubated for 90 minat room temperature and with gentle agitation in 10 ml of an antibodysolution containing a 1:5,000 dilution of the primary antibody (rabbitanti-PilA polyclonal antibodies raised against the 42 amino acids at thecarboxy-terminus of the PilA protein) and a 1:2,500 dilution of goatanti-rabbit IgG antibodies conjugated to the Cy^(tm) 5 fluorescence dye(ECL™ Plex, Amersham GE Healthcare). The membrane was washed 4 times (5min each) in wash buffer (TBS-T, pH 7.4, 0.1% Tween 20) and rinsed threetimes in wash buffer without Tween 20.

The protein bands that hybridized with the anti-PilA antibodies werevisualized after scanning the blot with a Typhoon imager (GE HealthcareSciences) operated in fluorescence mode (excitation at 633 nm, 670 BP 30filter, and PMT setting at 600 V).

Elemental Analyses

The SeqCHED web server (R. Levy, M. Edelman, V. Sobolev, Proteins 76,365 (Aug. 1, 2009) within the SPACE tools suite (V. Sobolev et al.,Nucleic Acids Res. 33, W39 (Jul. 1, 2005)) of the Weizmann Institute ofScience were used to identify conserved metal-ion binding sites in thetranslated gene sequence of the pilA gene (GSU 1496) of G.sulfurreducens. The amino acid sequence of the mature, processed pilin(starting at phenylalanine in position 11 (FIG. 2A), rather than thefull length precursor, was used for these analyses. Information aboutthis application and the metal-binding prediction algorithm can be foundat the website ligin.weizmann.ac.il/seqched/.

Quantitative elemental analysis of the purified pili preparations wasperformed by Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES) using a Thermo Jarrell-Ash Enviro 36 Inductively Coupled ArgonPlasm (Chemical Analysis Laboratory, University of Georgia, Athens). Forthese experiments, samples of purified pili (ca. 15 μg/ml) wereresuspended in 10 mM CHES buffer (pH 9.5, 1 mM EDTA) and analyzed inreference to blank control samples (same buffer without protein). TheLowest instrumental Limits of Detection (LOD) shown in Table 7 wereobtained from the CAIS website,www.uga.edu/cais/analytical_services/chemical_analysis/elements2.htm.The LOD values and measurements (in ppm) were used to calculate theamount of atoms per pilin subunit.

TABLE 7 Lowest Instrumental Limits of Detection (LOD) Elemental analysesof G. sulfurreducens LOD^(a) Pili elements pili by ICP-AES ppmatoms/pilin (atoms/pilin)^(b) Al 0.06 2.9 ± 0.9 <LOD Sb 0.09 6.9 ± 2.1<LOD As 0.08 3.3 ± 1.0 <LOD Ba 0.06 28.5 ± 8.6  <LOD Be 0.09 1.3 ± 0.4<LOD B 0.10 31 ± 9.3 <LOD Cd 0.06 3.8 ± 1.2 <LOD Ca 0.05 1.6 ± 0.5 <LODCr 0.06 3.1 ± 0.9 <LOD Co 0.06 3.6 ± 1.1 <LOD Cu 0.07 3.4 ± 1.0 <LOD Fe0.05 2.8 ± 0.8 <LOD Pb 0.06 39.5 ± 11.9 <LOD Mg 0.03 3.8 ± 1.1 <LOD Mn0.10 5.6 ± 1.7 <LOD Mo 0.05 1.6 ± 0.5 <LOD Ni 0.10 67.1 ± 20.2 <LOD P0.09 5.3 ± 1.6 <LOD K 0.50 9.0 ± 2.7 <LOD Se 0.09 0.9 ± 0.3 <LOD Si 0.502.3 ± 0.7 <LOD Ag 0.10 3.5 ± 1.1 <LOD Na 0.50 54.9 ± 16.5 <LOD Sr 0.051.8 ± 0.5 <LOD V 0.15 9.1 ± 2.7 <LOD Zn 0.05 2.4 ± 0.7 <LOD ^(a)Lowestinstrumental Limits of Detection (LOD) ^(b)Average value and standarddeviation of three biological replicates.

UV-VIS Absorption and Fluorescence Spectroscopy

Dry purified pili samples were resuspended in ½ volume of isopropanoland, then, ½ volume of ddH₂O. Standards with L-tyrosine, riboflavin andmenaquinone were also prepared as solutions in isopropanol and ddH₂O, asdescribed for the pili samples. Absorption spectra were collected with aCary100 UV-Vis spectrometer (Varian) set to 2 nm bandpass. Fluorescencespectra were measured with QuantaMaster spectrometer (Photon TechnologyInternational), with 270 nm excitation and 5 nm bandpass. All spectrawere collected at room temperature, in quartz cuvettes with 1 cm pathlength (Spectrocell Inc.)

Calculation of Fe(III) Oxide Respiratory Rates

Using rates of Fe(III) oxide reduction (measured as the production ofFe(II)) and cell growth (measured as number of cells from culturesdoubling every 15 h) reported in G. Reguera et al., Nature 435, 1098(Jun. 23, 2005), the electron transport rates per cell were inferred.From the linearity of Fe(II) production during the reduction of Fe(III)oxides, electron transport rates of 5×10¹² electrons per second werecalculated. After dividing this number by the culture's growth yield(5×10⁷ cells after the reduction of ca. 40 mM of Fe(III) oxides) arespiratory rate of ˜10⁵ electrons per cell per second was estimated.

Despite the absence of mediators, the protein matrix conducted electronsaxially and at rates several orders of magnitude above those measuredduring the respiration of Fe(III) oxides. These distances and ratesgreatly exceed the known limits for charge transport reactions throughprotein matrices and make the pilus nanowires a new paradigm in proteinelectron transfer.

Results

Electrostatic interactions between the pili during their purification atneutral pH resulted in thick bundles or ropes that did not solubilize inSDS and enabled their purification (FIG. 8A). These intermolecularinteractions were effectively destabilized at basic pH and enabled theseparation of the individual pilus fibers (FIG. 8B). The biochemicalcomposition of the pili was analyzed by fully depolymerizing the fiberswith octyl-glucoside and separated the pilus' protein components bydenaturing SDS-PAGE. The depolymerization yielded a single peptidesubunit with the apparent mass (ca. 6.5 kDa) expected for the maturePilA pilin and hybridized with anti-PilA antibodies (FIG. 8C). OmcS, a50-kDa outer membrane cytochrome which has been hypothesized in C.Leang. X. Qian, T. Mester, D. R. Lovley, Appl. Environ. MAicrobiol. 76,4080 (June, 2010) to adsorb to pili-like filaments in G. sulfurreducensand mediate conduction, was successfully removed during thepurification.

Despite the absence of proteins other than the PilA subunit, the piliwere conductive by scanning tunneling microscopy (STM) (FIGS. 7A-7C).STM imaging of the purified pili showed conducting filaments withperiodic molecular sub-structures, corresponding to regions of the pilusthat supply more tunneling current due to an increase in the localdensity of states (FIG. 7A).

FIG. 9 shows a STM topographical image (top) and height measurements(bottom) of a section of pilus fiber produced according to the methoddescribed in this Example and acquired in constant current mode (0.5V,100 pA). The height graph, at the bottom, shows a large (ca. 10 nm)apparent width of the pilus fiber (lower curve), due to the distortioncaused by the broadening effect of the tip as it crosses the pilus. Theaxial length measurements (upper curve) show voltage-dependent featuresevery 14 nm (upward pointing arrows) and topographical peaks every 2-3nm (downward pointing arrows). The axial periodicity included deep 14-nmrepeating electronic features interspersed with 3-4 nm periodictopographic substructures (FIG. 9). The apparent STM width was ca. 10 nm(FIG. 9), yet produced ca. 5 nm widths once the distortion caused by thebroadening effect of the tip while traversing the fiber was subtracted.

In contrast, pili purified from Pseudomonas aeruginosa strain K (PAK)were insulators in the ±1 V range and could only be imaged at largesample voltages (greater than 2 V) and low tunneling current set points(FIG. 7B). At these voltages, tip instabilities often result due to thelarge electric field between the tip and the sample, which causesdistortions and noise in the imaged data. I-V (current vs. voltage)measurements taken at fixed locations of the pilus filaments confirmedthe metallic (ohmic) behavior of the G. sulfurreducens pili atbiological (±1 V) voltages and the insulating behavior of the PAK pili(FIG. 7C).

FIGS. 10A and 10B show tunneling conductance, dI/dV, of the G.sulfurreducens pili (FIG. 10A) and the PAK pili controls (FIG. 10B)obtained as the numerical differentiation of the I and V values shown inFIG. 7C and plotted against the tip-sample bias voltage (V). As FIG. 10Ashows, the G. sulfurreducens pili produced a conductor-like spectrumwith electronic states at low voltages, never reaching zero conductance.Furthermore, the plot of the conductance, dI/dV, versus the tip-samplebias voltage, V, revealed clear electronic states at low voltages, neverreaching zero conductance in the G. sulfurreducens pili, which isconsistent with the behavior of a true conductor. This is in contrast tothe insulator-type spectrum of the PAK pili characterized by a large(±1.5-2 V) band gap at zero conductance (FIG. 10B). The STM analysesthus confirmed the unique electronic structure of the pili of G.sulfurreducens that enables them to function as electronic conduits.

The lack of proteins, other than the PilA pilin, in the purified piliexcluded any contribution from c-cytochromes to its conductivity but didnot exclude the possibility of metal mediators. Metals can bind toconserved structural protein motifs and mediate electron transferreactions while stabilizing the protein's secondary structure. Althoughthe predictive features of the SeqCHED server did not identify anyconserved metal-ion binding sites in the PilA peptide subunit, theassembly of pilins in the pilus shaft could create structural andsequence motifs for metal coordination. Thus, we analyzed the elementalcomposition of the pili by inductively coupled plasma-atomic emissionspectroscopy (ICP-AES).

Despite the high specificity and sensitivity of this technique at theidentification and quantification of trace elements, inorganic elementsknown to catalyze electron transfer (Fe, Cu, Mo) and redox catalysis (V,Mn, Fe, Co, Ni, Cu and W) in biological systems were below detectionlimits (Table 6). Low levels of Ca²⁺ were detected (1.7±0.9 atoms ofCa²⁺ per pilin subunit), consistent with the known affinity of purifiedpili for this cation as discussed in J. C. McMichael, J. T. Ou, J.Bacteriol. 138, 976 (June, 1979) and its role at neutralizing theelectrostatic interactions that promote pili aggregation (See L. Craiget al., Mol. Cell 23, 651 (2006)). This is in agreement with thebiological role of Ca²⁺ atoms at balancing charges in proteins asdiscussed in K. L. Haas, K. J. Franz, Chem. Rev. 109, 4921 (October,2009).

Flavin cofactors, such as flavin mononucleotide (FMN) or flavin adenosyldinucleotide (FAD), can also bind proteins and enable electron transferand redox reactions. The presence of flavins in the pilus protein wasinvestigated by UV-visible spectroscopy, based on the ability of theflavin's isoalloxazine ring to absorb light in the UV and visiblespectral range. Quinones, such as ubiquinones and menaquinones, functionas lipid soluble electron carriers between membrane-bound respiratorycomplexes, due to their ability to bind specific structural motifs inhydrophobic regions of quinone-reactive redox proteins. Type IV pili areanchored on the inner membrane of Gram-negative bacteria where they canaccept quinones from the membrane-bound menaquinone pool. They also havea narrow (6-11 Å) hydrophobic central channel that could potentiallyhouse quinones and create an internal pathway for electron transfer freeof solvents.

The purified pili absorbed strongly below 230 nm and at about 270 nm.FIG. 11A shows an absorption spectrum of the purified pili. The inset inFIG. 11A shows the same pili spectrum in comparison to riboflavin(dashed line), where the axes have been adjusted to scale.

FIG. 11B shows a fluorescence spectrum of the purified pili, where theinset shows fluorescence intensity as relative fluorescence unitscorrected by a factor of 10³ with L-tyrosine (solid line) andmenaquinone (dashed line) spectra. As with UV-VIS spectroscopy, thefluorescence emission from the pilin's tyrosines was used as a markerfor the pilus protein in reference to an L-tyrosine standard solution.The pili spectrum produced two single peaks at ca. 300 and 340 nm,corresponding to the excitation peaks of tyrosine and its ionized form,tyrosinate, respectively. Ionization of the phenolic hydroxyl group intyrosine is generated by a carboxylate group of a nearby aspartic orglutamic amino acid.

Note that tyrosines yield two fluorescence peaks corresponding to thetyrosine (Tyr) and tyrosinate (Tyr•) forms (FIG. 11B). These fluorescentspectra are consistent with the emission from peptide bonds and thepilin's tyrosine residues (FIG. 2A). Thus, the strong tyrosinate peakdetected in the pili spectrum reflects the contribution of neighboringacidic residues (3 aspartic and 2 glutamic residues in the pilin, asshown in FIG. 2A). Such emission is generally discussed in C. R. Cantor,P. R. Schimmel, Techniques for the study of biological structure andfunction. Biophysical Chemistry vol. 2 (W. H. Freeman, San Francisco,1980).

Notably, the pili spectra had no peaks in the visible region (at ˜360and ˜450 nm) where flavins absorb. These results demonstrate that thepili are not flavoproteins and do not co-purify with flavoproteins.Coincidentally, flavins can also fluoresce at 440-470 nm wavelengths orhigher (FIG. 11C), depending on the type of flavin cofactor and thenature of the flavin-binding site in the protein. However, thefluorescence spectrum of the G. sulfurreducens pili also had no peaksabove 390 nm (FIG. 11B (inset). Thus, despite their high quantum yieldcompared to tyrosines, flavins were not detected in the pili spectrum,providing additional, confirmatory evidence for the absence of flavinsin the pili.

The finding that the G. sulfurreducens pili are conductive in theabsence of other proteins and organic or inorganic cofactorsdemonstrates that the protein matrix is responsible for its conductance.Although electrons can travel through protein matrices, distance islimited to the <14 Å separation between the protein's redox centers thatis required for optimal electron tunneling. The observed transport ofelectrons across the ca. 5-nm pilus width (FIGS. 7A-7C) indicates thatthe pilus protein assembly could enable charge transport at greaterdistances.

To further investigate this possibility, we used CP-AFM to measure theaxial conductivity of individual pilus fibers deposited on goldelectrodes nanofabricated onto an insulating SiO₂ substrate. FIG. 12A isan AFM image of pili deposited onto a 25-nm thick gold electrodenanofabricated onto an insulating SiO₂ surface with the arrows pointingto an example where a pilus clearly overlaps the edge of the goldelectrode. FIG. 12B is a schematic of a two-point transport measurementbetween the gold electrode and a CP-AFM tip through a pilus filament.FIG. 12C show I-V (current-voltage) curves obtained with CP-AFM. Thecurve taken with the tip positioned on a pilus (red) was acquired at alocation about 200 nm from the gold electrode. In contrast, negligiblecurrent was detected with the same tip in contact with the substrate at100 nm from the gold edge (black). The inset shows data acquired withthe same tip in contact with the gold electrode, with the same verticaland horizontal units as the large plot.

The current was linear when the tip was positioned on a pilus filamentat 200-nm distances from the gold edge, while no current was detectedwhen the tip was positioned on the silicon substrate at 100-nm distancesfrom the gold edge. A resistance R=200 MΩ was measured along a 1 μm-longpilus and an electron transport rate of 3.1×10⁹ electrons per second ata potential of 100 mV. The measured resistivity (p, “rho”) was 0.4 Ω·cm.This number is less than half the lowest resistivity (1 Ω·cm) measuredfor Shewanella nanowires as discussed in M. Y. El-Naggar et al., Proc.Natl. Acad. Sci. USA 107, 18127 (Oct. 19, 2010), which rely onc-cytochromes for long-range electron transport as discussed in Y. A.Gorby et al., Proc. Natl. Acad. Sci. USA 103, 11358 (Jul. 25, 2006), andis even lower than moderately doped silicon nanowires (0.5 Ω·cm).

Based on the linearity of Fe(II) production and cell growth yieldsduring the reduction of Fe(III) oxides by G. sulfurreducens respiratoryrates of 10⁵ electrons per cell per second were calculated. These ratesare several orders of magnitude lower than the rates of electrontransport measured along the pilus. Thus, a single cell could dischargeall the respiratory electrons onto the Fe(Ill) oxides with only onepilus. Yet cells from Fe(III) oxides cultures display several (>10) pilion one side of the cell, consistent with a biological strategy thatmaximizes the redox active surface of the cell without limiting the rateof electron transfer. Not surprisingly, adaptively evolved strains of G.sulfurreducens with increased rates of extracellular electron transferalso are hyperpiliated and have a reduced outer membrane c-cytochromecontent.

These results demonstrate that G. sulfurreducens pili are proteinnanowires and catalyze electron transfer reactions at rates (˜10⁹electrons per second) that do not limit the respiratory rates of thecell during the reduction of Fe(III) oxides. Contribution from otherproteins and from organic and inorganic cofactors were ruled out, thussupporting a mechanism exclusively mediated by the pilus' proteinassembly. A solvent-free pathway through the pilus central channelmediated by quinones was not plausible because the conductive pili hadno detectable quinones. The hydrophobic nature of the pilus' innerchannel also prevents solvents from filling the pilus internal cavity,which is necessary for ionic conduction through an inner electrolyticchannel. Furthermore, elemental analyses by ICP-AES did not detect anyionic species such as Na⁺, K⁺, Ag⁺, Li⁺ and Cu⁺ that could havecontributed to ionic conduction in an electrolytic core or in the solidstate. Biologically-relevant metals, such as Fe, Cu, Mo, V, Mn, Co, Ni,and W, which are involved in biological electron transfer or redoxprocesses, or metal dopants, such as B and Cd, which are commonly usedto metalize insulating materials and inorganic nanowires were also belowthe limits of detection.

The ICP-AES technique used in this study had the sensitivity to detectseveral atoms of metals per pilin (Table 7). With an estimated distanceof 10.5 Å between pilin heads in the pilus shaft, it would have taken atleast one metal atom per assembled subunit to maintain optimal (<14 Å)tunneling distances. Yet despite the lack of metals, the pilus nanowiresdisplayed metallic-like properties.

The ability of the pilus protein matrix to transport electrons overdistances that greatly exceed the theoretical and experimental tunnelinglimits supports the involvement of multiple pathways such as multisteptunneling (hopping), single-step superexchange tunneling pathways and/orperhaps yet to be discovered transport mechanisms facilitated by theunique structural features of the Geobacter pilins and their assembly.The Geobacter pilin structure is divergent, as they lack the conservedC-t globular head of other bacterial pilins, such as the PAK pilins.This divergent structure may be adaptive and evolved in order to use thepili as electronic conduits. Furthermore, despite the structuralconservation of the al domain (the conserved N-t 53-residue long of theα-helix that promotes hydrophobic interactions between the assembledpilins), amino acid conservation in the Geobacter pilins is restrictedto the first 30-40 amino acids. These divergent amino acids could bepositioned at optimal distances in the pilus shaft so as to providepathways for electron transfer. Alternatively, these Geobacter-specificamino acids could affect subunit contacts during assembly, therebyaffecting the pilus flexibility and mechanical properties so as tofacilitate collision-exchange mechanisms and the electronic coupling ofredox-active amino acids required for fast charge transport

The finding that G. sulfurreducens pili are protein nanowires issignificant for nanotechnological applications. The potential tocustomize the functional properties of proteins via well-establishedgenetic engineering approaches far exceeds available methods for thefunctionalization of carbon nanotubes and inorganic nanowire surfaces.Furthermore, the conservation of the al assembly domain in the Geobacterpilins raises the possibility of mass-producing these nanowires bymolecular self-assembly in a cell-free environment. This contrasts withfabrication methods for other types of nanowires, which involve hightemperatures, toxic solvents, vacuums, and expensive equipment. Proteinnanowires also circumvent major concerns regarding cytotoxicity andgenotoxicity that limit commercial applications of carbon, metal, andmetal-oxide based nanoparticles, making them suitable for thedevelopment of biodegradable and biocompatible nano-electronic devicesand expanding the known applications of metallic nanowires.

Example 7 Yield of Nanowires Expressed by Geobacter sulfurreducens

To determine yields, cultures were grown at 25° C. according to themethods described in the above examples to induce the expression ofnanowires. Cultures were also grown at 30° C. to produce otherwiseidentical cells, but without the nanowires.

The resulting cells were lysed and total cell protein was calculated forsamples having a similar OD at approximately 600 nm, thus similar numberof cells.

Although the expression of the nanowires changed, the total cell proteindid not substantially change in these two cultures. For this reason, the30° C. protein value was subtracted from the 25° C. value, with theresulting value used as the amount of protein corresponding to thenanowire in the starting culture. Protein assays to the purifiednanowires resulting from the 25° C. culture were also performed.

The resulting yield was approximately 63%. It is likely that yields canbe even higher with improved methods or with more accurate assays ofpili production in the starting cultures.

Example 8 Molecular Rectifying Behavior of Purified Microbial Nanowires

Unless otherwise stated, materials and methods were as described inExample 5. Prior to analyses the dry purified pili were resuspended inddH₂O with 1 mM EDTA to remove trace elements.

The molecular rectifying behavior of purified microbial nanowires wasdemonstrated using scanning tunneling microscopy (STM). By probing thenanowire with a sharp tip, this electronic information can be resolvedatom-by-atom. In addition to atomic scale images of the material, STMcan monitor the tunneling current at a particular location as a functionof the applied voltage. This information is directly related to theelectronic density of states. A material with a high density of statesacts as an electronic conductor. Likewise, a material with a low densityof states acts like an insulator. In contrast, a rectifier, or diode, isan electronic component that acts as either a conductor or an insulator,depending on the sign of the voltage. The resulting action is thatcurrent can only flow in one direction. FIG. 13 illustrates the densityof states for an ideal rectifier.

This example provides reproducible measurements showing rectifyingbehavior on the edges of microbial nanowires. In this example, cells ofthe bacterium Geobacter sulfurreducens were grown undernanowire-inducing conditions (incubation at 25° C.). The nanowires werepurified as described in Example 5 and resuspended in ddH₂O in 1 mMEDTA. The purified nanowires were composed of a single peptide subunitthat polymerizes via hydrophobic interactions to make the nanowirefilament. The nanowires were adsorbed and air-dried onto a graphitesurface prior to STM analyses. The STM tip was positioned on thenanowire to acquire electronic information.

FIG. 14 includes a STM topographical image (left) of a purified nanowiredeposited onto a graphite surface together with three spectroscopicmeasurements of the electronic density of states (right). The top andbottom curves on the right panel were acquired at the top edge andbottom edge, respectively, at the locations indicated by the “+” symbolson the left image. The middle curve was acquired at the central “+”symbol. The blue arrows indicate enhanced density of states. The keyfeatures are highlighted by the arrows, which indicate enhanced densityof states near ±0.4 V. As the enhancement depends on the sign of thevoltage (i.e., the curves are asymmetric with respect to the horizontalaxis), the overall behavior at low voltages, in the range of ±0.8 V, isconsistent with a rectifier.

Although the STM measurements show the density of states in thetransverse direction (lateral conductivity), this behavior is consistentwith rectifying behavior in the longitudinal direction (axialconductivity). This is illustrated schematically in FIG. 15. FIG. 15(left) shows a likely charge path in the purified nanowire of FIG. 14.If the microbial nanowires were ohmic conductors, the plot would havebeen symmetrical. The enhanced density of states on opposite edges isconsistent with rectifying behavior in the axial direction, given thelikely helical path of current flow along the pilus.

The purified nanowires used in this example lack metals and are notassociated with any redox protein or cofactor, as discussed above inExample 5 (and Example 6). Thus, the molecular rectifying behavior ofmicrobial nanowires is a consequence of the biochemical nature of thenanowire (protein amino acid composition, structure, and chemicalmodifications). Because of this, genetic engineering can be used tomodify the native rectification properties to produce customizedrectifiers with electronic properties suitable for each particularapplication. The proteinaceous nature of these microbial rectifiers alsomakes them biodegradable and desirable for applications in nanomedicine.Furthermore, by using mass-production in recombinant hosts and in vitroassembly also the costs associated with nanowire and nanomedicinesynthesis can be reduced. Additional testing will include two-probe andfour-probe devices.

REFERENCES

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All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby specifically incorporated by reference to the same extent asif it had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and the methods and processes are not necessarilyrestricted to the orders of steps indicated herein or in the claims. Asused herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a nucleic acid” or “apolypeptide” includes a plurality of such nucleic acids or polypeptides(for example, a solution of nucleic acids or polypeptides or a series ofnucleic acid or polypeptide preparations), and so forth. Under nocircumstances may the patent be interpreted to be limited to thespecific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims and statements of theinvention.

The following statements of the invention are intended to describe andsummarize various embodiments of the invention according to theforegoing description in the specification.

STATEMENTS DESCRIBING EMBODIMENTS OF THE INVENTION

-   -   1. An isolated Geobacteraceae nanowire polypeptide that is        genetically or chemically modified.    -   2. The nanowire polypeptide of statement 1, which is genetically        modified and has at least 70% amino acid sequence identity to        any of SEQ ID NO:1-10.    -   3. The nanowire polypeptide of statement 1 or 2, which is        genetically modified and does not have a sequence that is        identical to an unmodified sequence selected from the group        consisting of SEQ ID NO:1-10.    -   4. The nanowire polypeptide of any of statements 1-3, which has        at least one amino acid replaced with a hydrophilic or aromatic        amino acid.    -   5. The nanowire polypeptide of any of statements 1-4, which has        at least one amino acid replaced with a tyrosine or tryptophan        residue.    -   6. The nanowire polypeptide of any of statements 1-5, which has        at least one amino acid replaced with a charged amino acid        residue.    -   7. The nanowire polypeptide of any of statements 1-6, which has        at least one amino acid replaced with an aspartic acid, glutamic        acid, lysine or arginine residue.    -   8. The nanowire polypeptide of any of statements 1-7, wherein at        least one hydrophobic or apolar amino acid is replaced with a        hydrophilic or aromatic amino acid.    -   9. The nanowire polypeptide of any of statements 1-8, wherein        the nanowire polypeptide secondary structure is substantially        helical.    -   10. The nanowire polypeptide of any of statements 1-9, wherein        at least 50% of the nanowire polypeptide's secondary structure        is α-helical.    -   11. The nanowire polypeptide of any of statements 1-10, wherein        at least 60% of the nanowire polypeptide's secondary structure        is α-helical.    -   12. The nanowire polypeptide of any of statements 1-11, wherein        at least 70% of the nanowire polypeptide's secondary structure        is α-helical.    -   13. The nanowire polypeptide of any of statements 1-12, wherein        the nanowire polypeptide assembles into a pilus.    -   14. The nanowire polypeptide of any of statements 1-13, wherein        the pilus is electrically conductive.    -   15. The nanowire polypeptide of any of statements 1-14, wherein        the nanowire polypeptide contains no metals.    -   16. The nanowire polypeptide of any of statements 1-15, wherein        the genetic or chemical modification modulates the conductive,        adhesive or coupling property of the nanowire polypeptide.    -   17. An isolated nucleic acid encoding the nanowire polypeptide        of any of statements 1-16.    -   18. The isolated nucleic acid of statement 17, comprising        nucleic acid sequence SEQ ID NO:11.    -   19. The isolated nucleic acid of statement 17 or 18, which is        incorporated into a replication or expression vector.    -   20. The isolated nucleic acid of statement 17, 18 or 19, which        is operably linked to an expression control sequence.    -   21. The isolated nucleic acid of statement 19 or 20, wherein the        vector further comprises a polyadenylation or transcriptional        termination sequence.    -   22. An isolated host cell comprising the isolated nucleic acid        of any of statements 1-21.    -   23. The isolated host cell of statement 22, comprising a pilT        gene and/or a pilB gene from a Geobacteraceae bacterium.    -   24. The isolated host cell of statement 22 or 23, wherein the        pilT gene and/or the pilB gene is from a Geobacter species.    -   25. The isolated host cell of any of statements 22-24, wherein        the cell is a prokaryotic or eukaryotic cell.    -   26. The isolated host cell of any of statements 22-25, wherein        the cell is a prokaryotic cell.    -   27. The isolated host cell of statement 25 or 26, wherein the        prokaryotic cell is a gram negative bacterium.    -   28. The isolated host cell of statement 25 or 26, wherein the        prokaryotic cell is a Geobacteraceae bacterium.    -   29. The isolated host cell of any of statements 25-28, wherein        the prokaryotic cell is Geobacter sulfurreducens.    -   30. The isolated host cell of any of statements 25-29, wherein        the prokaryotic cell is Geobacter sulfurreducens strain PCA.    -   31. The isolated host cell of any of statements 25-30, wherein        the host cell does not have a pilT gene.    -   32. The nanowire polypeptide of statement 1, which is chemically        modified to modulate the conductive, adhesive or coupling        properties of the nanowire polypeptide.    -   33. The nanowire polypeptide of statement 32, which is        chemically modified using a reagent selected from the group        consisting of performic acid, a peroxide, iodoacetamide,        iodoacetic acid, bissulfosuccinimidyl suberate,        1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,        N-ehtylmaleimide, or methyl methanethiosulfonate and        S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl        methanesulfonothioate (MTSL).    -   34. A pilus comprising the nanowire polypeptide of any of        statements 1-33.    -   35. A nanowire pilus comprising a protein filament isolated from        a bacterium, the filament comprising the nanowire polypeptide of        any of statements 1-33 as peptide subunits capable of assembling        into the protein filament and capable of establishing an        electrical connection with an insoluble electron acceptor.    -   36. A rectifier comprising: one or more genetically or        chemically modified nanowire polypeptides of any of statements        1-16 in a pilus capable of establishing an electrical connection        with an insoluble electron acceptor.    -   37. The rectifier of statement 36, wherein the genetically or        chemically modified nanowires have substantially the same amino        acid sequence.    -   38. The rectifier of statement 36, wherein at least one of the        genetically or chemically modified nanowires has a different        amino acid sequence from other nanowires in the pilus.    -   39. The rectifier of any of statements 36-38 wherein the        insoluble electron acceptor is selected from Fe(III) oxide        minerals, an electrode, a second isolated pilus or combinations        thereof.    -   40. The rectifier of any of statements 36-39 adapted for use in        radio demodulation, low voltage AC-DC power conversion, current        steering, power switches, over voltage protection, logic        circuitry in electronic devices or computer chips.    -   41. The rectifier of any of statements 36 to 40 capable of        functioning as an asymmetric conductor for voltages having a        range of ±1.2 V.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

Other embodiments are described within the following claims.

1. An isolated Geobacteraceae nanowire polypeptide that has at least 95%amino acid sequence identity to any of SEQ ID NO: 1-10.
 2. The nanowirepolypeptide of claim 1, which is genetically modified and does not havea sequence that is identical to an unmodified sequence selected from thegroup consisting of SEQ ID NO:1-10.
 3. The nanowire polypeptide of claim1, wherein at least one hydrophobic or apolar amino acid is replacedwith a hydrophilic or aromatic amino acid.
 4. The nanowire polypeptideof claim 1, which has at least one amino acid replaced with a tyrosineor tryptophan residue.
 5. The nanowire polypeptide of claim 1, which hasat least one amino acid replaced with an aspartic acid, glutamic acid,lysine or arginine residue.
 6. The nanowire polypeptide of claim 1,wherein at least 50% of the nanowire polypeptide's secondary structureis α-helical.
 7. The nanowire polypeptide of claim 1, wherein thenanowire polypeptide assembles into an electrically conductive pilus. 8.The nanowire polypeptide of claim 1, wherein the nanowire polypeptidecontains no metals. 9-19. (canceled)
 20. The nanowire polypeptide ofclaim 1, which is chemically modified to modulate the conductive,adhesive or coupling properties of the nanowire polypeptide.
 21. Thenanowire polypeptide of claim 1, which is chemically modified using areagent selected from the group consisting of performic acid, aperoxide, iodoacetamide, iodoacetic acid, bissulfosuccinimidyl suberate,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-ehtylmaleimide, ormethyl methanethiosulfonate andS-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methylmethanesulfonothioate.
 22. A nanowire polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 1-10,wherein the amino acid sequence is genetically or chemically modified sothat the nanowire polypeptide has electrical conductivity activity thatis less than 90% or greater than 120% of the electrical conductivityactivity of a wild type nanowire polypeptide comprising SEQ ID NO: 1-10.23. A pilus comprising the nanowire polypeptide of claim
 1. 24-28.(canceled)
 29. A mutated nanowire polypeptide comprising the amino acidsequence of SEQ ID NO: 9 or 10, wherein at least one hydrophobic orapolar amino acid selected from the group consisting of A11, I12, I13,I15, L16, A17, A18, I19, A20, I21, P22, A26, V29, A31, A35, A36, I40,L43, A46, L47, A50, A52, P58 and P59 is substituted or replaced with ahydrophilic or aromatic amino acid to modify electrical conductivity.30. The mutated nanowire polypeptide of claim 29, wherein the encodedpolypeptide has at least one amino acid replaced with a tyrosine ortryptophan residue.
 31. The mutated nanowire polypeptide of claim 29,wherein the encoded polypeptide has at least one amino acid replacedwith an aspartic acid, glutamic acid, lysine or arginine residue. 32.The mutated nanowire polypeptide of claim 29, wherein the encodedpolypeptide has at least 50% of the polypeptide's secondary structure asα-helix.
 33. The mutated nanowire polypeptide of claim 29, wherein theencoded polypeptide assembles into an electrically conductive pilus.